Methods and kits for stimulating production of megakaryocytes and thrombocytes

ABSTRACT

The invention provides methods for stimulating thrombocytopoiesis in a mammal comprising administering to the mammal an effective amount of modified C-reactive protein (&#34;mCRP&#34;) or mutant protein expressing neo-CRP antigenicity. Methods for treating thrombocytopenia, and for promoting megakaryocyte growth and maturation in vitro are also provided. Kits and articles of manufacture are further provided which include mCRP or mutant protein expressing neo-CRP antigenicity.

This application is a continuation in part of application Ser. No.08/202,033, filed Feb. 23, 1994, now U.S. Pat. No. 5,547,931.

FIELD OF INVENTION

The invention relates to methods of stimulating megakaryocytopoiesis andthrombocytopoiesis with modified C-reactive protein ("mCRP") and mutantprotein expressing neo-CRP antigenicity. The invention also relates tomethods of treating thrombocytopenia, and to kits containing mCRP andthe mutant protein.

BACKGROUND OF THE INVENTION

1. CRP Structure and Activity

C-reactive protein was first described by Tillett and Francis [J. Exp.Med., 52:561-71 (1930)] who observed that sera from acutely ill patientsprecipitated with the C-polysaccharide of the cell wall of Streptococcuspneumonia. Other investigators subsequently identified the reactiveserum factor as protein, hence the designation "C-reactive protein" or"CRP." Kilpatrick et al., Immunol. Res., 10:43-53 (1991), provides arecent review of CRP.

CRP is a pentameric molecule which consists of five identical subunits[Osmand et al., Proc. Natl. Acad. Sciences, U.S.A., 74:739-743 (1977)].This pentameric form of CRP is sometimes referred to as "native CRP."

The gene sequence for human CRP has been cloned [Lei et al., J. Biol.Chem., 260:13377-13383 (1985)]. In addition, the primary sequences forrabbit CRP [Wang et al., J. Biol. Chem., 257:13610-13615 (1982)] andmurine CRP have been reported [Whitehead et al., Biochem. J.,266:283-290 (1990)], and is under investigation for rat, dog, horse,goat, and sheep. Clinical and laboratory observations have determinedthat the acute phase response, classically defined by the well-definedchanges of the blood [Pepys et al., Advances in Immunology, 34:141-212(1983)], develops during various states of disease and injury includingmalignant neoplasia, ischemic necrosis, and bacterial, viral, or fungalparasitic infections. Measurement of serum acute phase reactants such asCRP have been utilized in clinical tests for diagnosis and clinicalmanagement of patients with various conditions, including systemic lupuserythematosus (SLE) [Bravo et al., J. Rheumatology, 8:291-294 (1981)],rheumatoid arthritis [Dixon et al., Scand. J. Rheumatology, 13:39-44(1984)], graft versus host disease [Walker et al., J. Clin. Path.,37:1022-1026 (1984)], as well as many other diseases.

2. Modified-CRP Structure and Activity

In about 1983, another form of CRP was discovered which is referred toas "modified C-reactive protein" or "mCRP." mCRP has significantlydifferent charge, size, solubility and antigenicity characteristics ascompared to native CRP [Potempa et al., Mol. Immunol., 20:1165-75(1983)]. mCRP also differs from native CRP in its bindingcharacteristics. For instance, mCRP does not bind phosphorylcholine[Id.; Chudwin et al., J. Allergy Clin. Immunol., 77:216a (1986)].

The distinctive antigenicity of mCRP has been referred to as "neo-CRP."Neo-CRP antigenicity is known to be expressed on:

1) CRP treated with acid, urea or heat under certain conditions;

2) the primary translation product of DNA coding for human and rabbitCRP; and

3) CRP immobilized on plastic surfaces [Potempa et al., Mol. Immunol.,20:1165-75 (1983); Mantzouranis et al., Ped. Res., 18:260a (1984);Samols et al., Biochem. J., 227:759-65 (1985); Potempa et al., Mol.Immunol., 24:531-541 (1987)]. A molecule reactive with polyclonalantibody specific for neo-CRP has been identified on the surface of10-25% of peripheral blood lymphocytes (predominantly NK and B cells),80% of monocytes, and 60% of neutrophils, and as well as at sites oftissue injury [Potempa et al., FASEB J., 2:731a (1988); Bray et al.,Clin. Immunol. Newsletter, 8:137-140 (1987); Rees et al., Fed. Proc.,45:263a (1986)].

Furthermore, mCRP differs from native CRP in its biological activity. Ithas been reported that mCRP can influence the development of monocytecyto-toxicity, improve the accessory cell function of monocytes,potentiate aggregated IgG-induced phagocytic cell oxidative metabolism,and increase the production of interleukin-1, prostaglandin E andlipoxygenase products by monocytes [Potempa et al., Protides Biol.Fluids, 34:287-290 (1987); Potempa et al., Inflammation, 12:391-405(1988); Chu et al., Proc. Amer. Acad. Cancer Res., 28:344a (1987);Potempa et al., Proc. Amer. Acad. Cancer Res., 28:344a (1987); Zeller etal., Fed. Proc., 46:1033a (1987); Chu et al., Proc. Amer. Acad. CancerRes., 29:371a (1988)].

In vivo experiments with mCRP were performed to determine if mCRP wascapable of providing a protective effect against lethal doses ofStreptococcal pneumonia [Chudwin et al., J. Allergy Clin. Immunol.,77:216a (1986)]. These studies demonstrated that intravenousadministration of mCRP not only protected the animals from lethal S.pneumonia doses but that mCRP efficacy was 3 to 4 fold greater thannative CRP.

3. Hematopoietic Cell Production and Activity

Pluripotent stem cells in the bone marrow of mammals have the potentialto give rise to different types of blood cells which circulate in theperipheral blood. The pluripotent stem cells differentiate into variouscell lineages through multiple maturational stages, thereby giving riseto committed blood cell types.

One cell lineage differentiated in the bone marrow is the megakaryocyticlineage. The earliest recognizable member of the megakaryocytic lineageis the megakaryoblast. A morphologic classification commonly applied tothe megakaryocyte lineage refers to the megakaryoblast as the earliestcell form, the promegakaryocyte or basophilic megakaryocyte as theintermediate cell form, and the mature megakaryocyte as the final cellform. The mature megakaryocyte then forms and extends filaments ofcytoplasm (also called pseudopods) that detach and fragment intoindividual thrombocytes or platelets. The process of thrombocyteformation, starting from the earliest blast cell stage, typically takesabout three days.

Thrombocytes generally circulate in the peripheral blood and play animportant role in the body's response to injury or trauma. For example,thrombocytes can become activated and aggregate at the site of injury ortrauma. Thrombocytes are also secretory cells, containing variousgranules which are the secretory organelles of the cell. Such granulesinclude alpha granules, dense granules, and lysosomal granules[Harrison's Principles of Internal Medicine, 9th Ed., McGraw-Hill, NewYork, 1980].

Alpha granules are typically the first granules released fromthrombocytes. The alpha granules contain several proteins, includingplatelet factor 4, beta-thromboglobulin, and fibrinogen. The densegranules are usually released after the alpha granules. The densegranules contain calcium, ADP, serotonin, and catecholamines. Thelysosomal granules are usually released last and contain enzymes such asphosphatase, beta-glucuronidase, and cathespin. These proteins andenzymes are released from the thrombocyte granules in response tocertain stimuli and assist in potentiating thrombus, or blood clot,formation.

The effects of native CRP and altered forms of CRP on the activation,aggregation, and secretory function of circulating thrombocytes has beenthe subject of several reports. Fiedel et al., Immunology, 45:439-447(1982), describe that thermally-aggregated CRP induced isolatedplatelets to aggregate and secrete in in vitro culture. Fiedel et al.also discuss the ability of aggregated CRP to initiate plateletresponsiveness and enhance platelet activation in plasma stimulated byvarious platelet agonists [See also, Fiedel et al., Clin. Exp. Immunol.,50:215-22 (1982)].

Miyazawa et al., J. Immunol., 141:570-74 (1988), report that FA-CRP(defined as human CRP treated with an Fe² +-ascorbate) in combinationwith suboptimal doses of platelet-activating factor and other stimulatoragents activated platelets in vitro. The authors also observed that theFA-CRP did not show activity toward rabbit platelets, and therefore,concluded that the activity was species specific.

Fiedel, Blood, 65:264-69 (1985), disclose that aggregated human CRP incombination with suboptimal concentrations of ADP in platelet-richplasma induced platelets to aggregate, secrete dense and alpha-granuleconstituents, and generate thromboxane A₂.

Potempa et al., Inflammation, 12:391-405 (1988), disclose that mCRP iscapable of activating platelets, PMNL, and monocytes in vitro. Theauthors also report that in certain culture conditions, mCRP activatedplatelets to aggregate.

Other investigations have focused on examining the processes by whichthrombocytes are made and the factors which influence those processes.The production of thrombocytes has typically been viewed as a processinvolving two different stages. The first stage is directed toproliferation or differentiation of megakaryocytes. The second stage isdirected to maturation or release of the megakaryocytes intothrombocytes. A review of thrombocytopoiesis and megakaryocytopoiesis isprovided in The Platelet, an International Academy of PathologyMonograph, published by The Williams & Wilkins Company, 1971.

It is generally recognized that different factors are needed for eachstage of the production of thrombocytes [See, e.g., Murphy, Hematol.Oncol. Clin. North Am., 3:465-478 (1989)]. A first factor is an inducerof the proliferation or clonal growth of megakaryocyte progenitors. Thisfactor is sometimes referred to as Megakaryocyte Colony StimulatingFactor (Meg-CSF). A second factor is a promoter of maturation ofmegakaryocytes and formation and release of platelets. This factor issometimes referred to as a Megakaryocyte-Potentiator (Meg-POT) factor.Factors which exhibit Megakaryocyte-potentiator activity typically havethe ability to promote megakaryocyte colony formation in the presence ofMeg-CSF, to stimulate megakaryocyte polyploidization, and induce thematuration of megakaryocytes.

Thrombocytopoiesis and megakaryocytopoiesis may be adversely affected bydifferent diseases or pathological conditions. Cell production may alsobe adversely affected by radiation, drugs, or surgery. While therapiessuch as surgery, chemotherapy, and radiation have improved, they arenonetheless often accompanied by damage to bone marrow and/or otherblood cell-producing tissues.

In diagnosing such conditions or monitoring the effects of certaintherapies, platelet counts in the peripheral blood are typicallymeasured. A decrease in the number of platelets in the blood can occurin certain medical disorders [Marchasin et al., California Medicine,101:95-100 (1964)]. Thrombocytopenia, a medical condition characterizedby a low platelet count in the blood, can result from impairedproduction of platelets by the bone marrow, platelet sequestration inthe spleen, or increased destruction of platelets in the peripheralcirculation. Further, for patients receiving large volumes of rapidlyadministered platelet-poor blood products, thrombocytopenia can developdue to dilution of the blood.

In addition to measuring platelet numbers, platelet volume, alsoreferred to as mean platelet volume ("MPV"), can be measured. Anincrease in MPV has been associated with increased megakaryocyte size inresponse to thrombopoietic stress [Thompson et al., Blood, 72:1 (1988)].It has also been observed that platelet count and MPV are inverselyrelated, i.e., patients with low platelet counts tend to have larger MPVand patients with high platelet counts tend to have smaller MPV [Id.].This inverse relationship has been interpreted as one mechanism by whichthe body maintains a relatively stable platelet mass. The "plateletmass" is determined by multiplying the platelet number by the meanplatelet volume.

Thrombocytopenia and other conditions characterized by abnormal plateletnumbers or volume have been treated in various ways. One method employedto treat thrombocytopenia is platelet transfusion. Platelet transfusionscan be effective in some circumstances, but are undesirable because ofthe costs and risk of infections.

Certain cytokines, humoral factors, and chemical compounds have beenidentified as inducing thrombocyte production and megakaryocyte growth[See, for example, U.S. Pat. No. 5,126,325 (human B cell differentiationfactor); U.S. Pat. No. 5,032,396 (interleukin-7); U.S. Pat. No.5,250,732 (ketamine analogues); U.S. Pat. No. 5,260,417 (megakaryocytegrowth promoting factor, "MGPA")]. In vitro, interleukin-3 (IL-3),interleukin 6 (IL-6) interleukin-11 (IL-11) and granulocyte-macrophagecolony-stimulating factor (GM-CSF) have been reported to increase thenumber and size of megakaryocyte colonies [Williams, Immunol. Ser.,49:215-229 (1990); Hoffman et al., Yale J. Biol. Med., 63:411-418(1990); Yonemura et al., Exp. Hematol, 20:1011 (1992); Teramura et al.,Blood, 79:327 (1992); Carrington et al., Blood, 77:34 (1991)]. IL-3 isbelieved to principally affect the differentiation (earliest) phase ofthe thrombopoiesis process [Moore et al., Blood, 78:1 (1991); Sonoda etal., PNAS USA, 85:4360 (1988)]. In contrast, IL-11 has been reported toinfluence thrombopoiesis principally at the maturation (later) phase[Teramura et al., Blood, 79:327 (1992); Yonemura et al., Exp. Hematol.,20:1011 (1992)].

Several investigators have reported that in vivo administration ofinterleukin-6 increased thrombocyte counts in the peripheral blood ofprimates [Asano et al., Blood, 75:1602 (1990)] and mice [Podja et al.,Exp. Hematol., 18:1034 (1990); Ishibashi et al., Blood, 74:1241 (1989)].Carter et al. reported that administration of exogenous thrombopoietindecreased the severity and duration of radiation-inducedthrombocytopenia in mice [Radiation Research, 132:74-81 (1992)].

SUMMARY OF THE INVENTION

The invention provides a method of stimulating thrombocytopoiesis in amammal comprising administering to the mammal an effective amount ofmodified C-reactive protein or mutant protein expressing neo-CRPantigenicity in a pharmaceutically-acceptable carrier.

The invention also provides a method of treating thrombocytopeniacomprising administering to a mammal diagnosed as havingthrombocytopenia an effective amount of modified C-reactive protein ormutant protein expressing neo-CRP antigenicity in apharmaceutically-acceptable carrier.

The invention also provides a method for promoting maturation ofmegakaryocytes in vitro. The method comprises the steps of providinghematopoietic cells in a cell culture medium and culturing the cells inthe presence of an effective amount of modified C-reactive protein ormutant protein expressing neo-CRP antigenicity.

The invention further provides an article of manufacture and kitcontaining materials useful for stimulating thrombocytopoiesis andmegakaryocytopoiesis. The article of manufacture comprises a container,a label on the container, and a composition contained within thecontainer, the composition being effective for stimulatingthrombocytopoiesis and megakaryocytopoiesis, and the label on thecontainer indicating that the composition can be used for stimulatingthrombocytopoiesis and megakaryocytopoiesis. The active agent in thecomposition comprises modified C-reactive protein or mutant proteinexpressing neo-CRP antigenicity. The kit comprises the container holdingthe composition effective for stimulating thrombocytopoiesis andmegakaryocytopoiesis, as well as other compositions such as buffers anddiluents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram of a series of polymerase chain reactions used toproduce a recombinant mutant protein expressing neo-CRP antigenicity.

FIG. 1B is a restriction map of plasmid pIT4.

FIGS. 2A and 2B illustrate the preparation of plasmid pIT3.

FIG. 3 is a photograph of an SDS-PAGE gel which includes samples ofrecombinant mutant protein.

FIGS. 4A-4C are graphs of the results of ELISA assays to detect thepresence of native CRP and mCRP antigenic determinants on recombinantmutant protein.

FIG. 5A is a bar graph showing the effect of mCRP on megakaryocytecolony formation in vitro.

FIG. 5B is a graph of the effect of mCRP on the kinetics ofmegakaryocyte colony formation in vitro.

FIGS. 6A and 6B are photographs of control and mCRP-treated murinemegakaryocyte colonies.

FIGS. 7A and 7B are graphs showing the effect of mCRP (subcutaneouslyinjected) on thrombocytopoietic activity in normal mice.

FIG. 7C is a graph showing the effect of mCRP (intraperitoneallyinjected) on thrombocytopoietic activity in normal mice.

FIGS. 8A and 8B are graphs showing the effect of mCRP on platelet countsin Cyclophosphamide-treated mice.

FIGS. 8C and 8D are graphs showing the effect of mCRP on platelet massin Cyclophosphamide-treated mice.

FIGS. 9A and 9B are graphs showing the effect of mCRP on platelet countsin X-irradiated mice.

FIGS. 9C and 9D are graphs showing the effect of mCRP on platelet massin X-irradiated mice.

FIGS. 10A-10E are graphs showing the effect of mCRP on platelet countsin HIV-positive humans.

FIGS. 11A and 11B are graphs showing the effect of mCRP on platelet massin HIV-positive humans.

FIG. 12 is a diagram of a series of polymerase chain reactions.

FIG. 13 illustrates the preparation of plasmid pIT13.

FIG. 14 presents representative SDS-PAGE results for the completepurification scheme for isolating a mutant protein according to theinvention from E. coli BLR(DE3) bearing plasmid pIT13.

FIG. 15A is an SDS-PAGE gel obtained by electrophoresing mCRP and amutant protein according to the invention purified from E. coli BLR(DE3)bearing plasmid pIT13.

FIG. 15B is the Western blot obtained by using anti-neo-CRP monoclonalantibody 3H12 to stain the SDS-PAGE electrophoretic patterns of mCRP anda mutant protein according to the invention purified from E. coliBLR(DE3) bearing plasmid pIT13.

FIGS. 16A-C are graphs of the results of ELISA assays performed todetect the presence of neo-CRP antigenic determinants on mCRP and amutant protein according to the invention purified from E. coli BLR(DE3)bearing plasmid pIT13. Three different anti-neo-CRP monoclonalantibodies were used: 3H12 (FIG. 16A); 8C10 (FIG. 16B); and 7A8 (FIG.16C).

FIGS. 17A-C are graphs showing the effects of mutant rCRP produced bypIT13 on murine thrombopoiesis.

FIGS. 18A-C are graphs showing the effects of mutant rCRP produced bypIT13 on murine thrombopoiesis.

FIG. 19 is a graph showing the effects of mCRP and mutant rCRP producedby pIT13 on murine thrombopoiesis.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The present invention provides methods for stimulatingmegakaryocytopoiesis and thrombocytopoiesis using modified C-reactiveprotein ("mCRP"). As discussed in the Background section, mCRP is amodified form of CRP which expresses neo-CRP antigenicity. Neo-CRPantigenicity is expressed on mCRP but not on native CRP.

The formation of mCRP involves the dissociation of native CRP into itssubunits. The formation of mCRP also involves a change in theconformation of the subunits. Although the subunits tend toself-aggregate [Potempa et al., Mol. Immunol., 24:531-41 (1987)], thesubunits in the aggregate form also express neo-CRP antigenicity.Accordingly, mCRP employed in the invention may consist of free subunitsand/or aggregates of subunits. Dissociated subunits do not reassembleinto native CRP; the change from native CRP to mCRP is irreversible[Potempa et al., Fed. Proc., 44:1190a (1985); Kresl et al., FASEB J.,5:633a (1991)].

The mCRP useful in the practice of the present invention can be preparedin a number of ways. For instance, the mCRP can be prepared using nativeCRP as a starting material. Methods of isolating native CRP from naturalsources are known in the art and are described, for example, byVolanakis et al., J. Immunol., 113:9-17 (1978); de Beer et al., J.Immunol. Meth., 50:17-31 (1982); Potempa et al., Mol. Immunol.,24:531-541 (1987). CRP is preferably isolated from serum, plasma, orpleural or ascites fluid by calcium-dependent affinity chromatographyusing phosphorylcholine-substituted BioGel® A 0.5 m (an agarose-basedresin obtained from BioRad Laboratories, Richmond, Calif.) [See, Potempaet al., Mol. Immunol., 24:531-541 (1987)]. This CRP isolation method isfurther described in Example 1 below. Using this isolation method, CRPcan be obtained which is about 99% pure. Partially purified CRP may alsobe obtained from commercial sources, such as Western States Plasma(Fallbrook, Calif.).

Methods of making mCRP from CRP are also known in the art [See, e.g.,Potempa et al., Mol. Immunol., 20:1165-1175 (1983); Potempa et al., Mol.Immunol., 24:531-541 (1987)]. For instance, mCRP can be prepared bydenaturing CRP. CRP can be denatured by treatment with an effectiveamount of urea (preferably 8M) in the presence of a conventionalchelator (preferably ethylenediamine tetraacetic acid (EDTA) or citricacid). Further, CRP can be treated to produce mCRP by adjusting the pHof the protein to below about 3 or above about 11-12. Finally, mCRP canbe produced by heating CRP above 50° C. for a time sufficient to causedenaturation (preferably at 63° C. for 2 minutes) in the absence ofcalcium or in the presence of a chelator. Methods of producing the mCRPfrom CRP using such techniques are also described further below inExample 1.

The mCRP prepared according to these methods may be from any species.There is substantial homology between the amino acid sequences of CRPfrom different species. For instance, there is from about 50% to about80k sequence homology between CRP from various mammalian species [Hu etal., Biochem., 25:7834-39 (1986); Whitehead et al., Biochem. J.,266:283-90 (1990); Kilpatrick et al., Immunol. Res., 10:43-53 (1991)].It is, therefore, expected that mCRP from any species will be effectivein the presently claimed invention. Thus, a mammal may be treated withmCRP from a different species (e.g., mice can be treated with humanmCRP). Alternatively, and preferably, the mammal is treated withhomologous mCRP (e.g., humans are treated with human mCRP) to avoidimmune reactions to the mCRP.

CRP and mCRP may also be prepared using genetic engineering techniquesand procedures of molecular biology. The primary translation product ofthe CRP gene (called preCRP, the CRP subunit with a presequence at theamino terminus) expresses neo-CRP antigenicity [Mantzouranis et al.,Ped. Res., 18:260a (1984)]. Accordingly, mCRP can be prepared byselecting conditions so that the CRP subunits are not assembled intopentameric native CRP in the host cell. This can be accomplished byexpressing the desired genomic or cDNA clone in a prokaryotic host [SeeSamols et al., Prot. Biol. Fluids, 34:263-66 (1986)]. The mCRP producedin this manner may consist of aggregates of CRP subunits and/or preCRPand perhaps other CRP peptides. See Id. This form of mCRP is insoluble,and further purification may be problematical. However, it should bepossible to inject this insoluble material into mammals as a suspensionwithout further processing, since an aggregated form of mCRP preparedfrom CRP purified from plasma has been shown to be effective, asdescribed further in the Examples below.

As used in the present invention, the term "mCRP" is intended to referto subunits of CRP, in free or aggregate form, which express neo-CRPantigenicity. It is believed that fragments of the CRP subunits may havethe same activities described herein for mCRP, and the use of suchfragments is considered to come within the scope of the presentinvention. It is also believed that proteins substantially homologous toCRP will have the activities described herein for mCRP, and suchproteins are also considered to come within the scope of the presentinvention.

The mCRP discussed above may be characterized and distinguished fromnative CRP on the basis of physiochemical properties, bindingcharacteristics or biological activities. As noted above, mCRP expressesneo-CRP antigenicity, whereas native CRP does not. Neo-CRP antigenicitycan be detected using polyclonal antisera specific for neo-CRP [See,Potempa et al., Mol. Immunol., 24:531-541 (1987)]. Preferably, however,mCRP is distinguished from native CRP using monoclonal antibodies likethose described in U.S. Pat. No. 5,272,257, the disclosure of which isincorporated herein by reference. Hybridomas secreting the monoclonalantibodies disclosed in U.S. Pat. No. 5,272,257 are deposited with theAmerican Type Culture Collection, Rockville, Md., and are registered asHB10175 (mAb 15.2C10), HB10176 (mAb 26.8C10), HB10177 (mAb 13.3H12), andHB10178 (mAb 15.1D6). These monoclonal antibodies are also described inYing et al., J. Immunol., 143:221-28 (1989). The antisera and antibodiescan be used, for example, in ELISA assays to distinguish mCRP fromnative CRP.

In addition, mCRP may be distinguished from native CRP on the basis ofcharge, solubility, binding characteristics and biological activity asreferenced in the Background section. The biological activities of mCRPinclude its ability to bind aggregated immunoglobulin and immunecomplexes which allows mCRP to be used to removed aggregatedimmunoglobulin and immune complexes from fluids (such as antibodyreagents or body fluids), to quantitate immune complexes, and to reducethe levels of immune complexes in a mammal in need thereof. However, toshow that a preparation contains mCRP, it is usually sufficient toestablish that the preparation 1) reacts positively with an antibodyspecific for an epitope found only on mCRP or 2) binds aggregatedimmunoglobulin (e.g., aggregated IgG).

The methods of the invention may alternatively employ a mutant proteinwhich expresses neo-CRP antigenicity. The mutant protein has at leastone amino acid added, deleted or replaced as compared to an unmutatedCRP subunit or unmutated preCRP. However, the mutant protein may haveseveral amino acid changes as compared to the unmutated CRP subunit orunmutated preCRP. For instance, the mutant protein may have severaladded amino acids, several deleted amino acids, several replacementamino acids, or a combination of added, deleted or replacement aminoacids, as compared to the unmutated CRP subunit or preCRP. Examples ofthe mutant protein of the invention are described in Example 2 below,and are referred to as "mutant rCRP's." Mutant proteins of the inventionare also described in co-owned and co-pending U.S. patent applicationSer. No. 08/296,545, filed Aug. 26, 1994.

The amino acid(s) added, deleted and/or replaced are chosen so that themutant protein retains neo-CRP antigenicity. The amino acid(s) can beidentified, for example, by using group-specific modification reactionslike those discussed in Chemical Modifications of Proteins, G. E. Meansand R. E. Feeney, Holden-Day, Inc. San Francisco, Calif. (1971) andChemistry of Protein Conjugation and Cross-linking, S. S. Wong, CRCPress Boca Raton, Fla. (1991). For instance, amino acid residues in aprotein having a free primary amine group such as is found in lysineresidues can have the primary amine group altered using variousanhydride agents such as acetic anhydride, succinic anhydride, or maleicanhydride. Accessible primary amine groups can also be modified usingreductive alkylation reactions with various aldehyde and ketone groups.Amino acid(s) and/or peptide regions which comprise selective antigenicepitopes specific for neo-CRP antibodies may also be defined by peptidemapping techniques as described in Ying et al., Mol. Immunol.,29:677-687 (1992).

It is preferable to choose such amino acid(s) addition, deletion, and/orreplacement so that the mutant protein is less likely to formnon-dissociable aggregates than the unmutated CRP subunit or unmutatedpreCRP. Suitable amino acid changes include the deletion or replacementof at least one, preferably all, of the cysteines in an unmutated CRPsubunit or unmutated preCRP. All CRP subunits and preCRP's contain atleast one cysteine. Mammalian CRP subunits contain two cysteines andmammalian preCRP's contain three cysteines. It is believed that some ofthese cysteines form intermolecular disulfide bonds, therebycontributing to the formation of non-dissociable cross-linkedaggregates. Therefore, one, two, or preferably all three, of thesecysteines are desirably deleted or replaced. When the cysteines arereplaced with other amino acids, they are preferably replaced withglycine, alanine, valine, leucine, isoleucine, serine, threonine ormethionine, but any amino acid can be used. Most preferred issubstitution with alanine.

As a result of the amino acid changes in them, the mutant proteins ofthe invention are easier to purify with much higher yields thanunmutated CRP subunits or unmutated preCRP's. (See, e.g., Example 2).Also, the final product is much purer with many fewer aggregates andfragments than that obtained with unmutated CRP subunits or unmutatedpreCRP's.

Not all of the amino acid additions, deletions and replacements needcontribute to the reduced likelihood of forming non-dissociableaggregates as long as the combined effect of all the changes is areduction in intermolecular non-dissociable cross-linking. For instance,the recombinant DNA manipulations used to produce the mutant proteinsmay result in amino acids being added at the amino or carboxy terminalends of the CRP subunit. This is acceptable as long as these amino acidsdo not contribute to the production of non-dissociable aggregates (See,e.g., Example 2). In addition, some of the amino acid changes may bemade for other purposes. For instance, it is desirable to make aminoacid changes which increase the solubility of the resultant mutantprotein in aqueous media, since a more soluble mutant protein is easierto purify and process. It has been found that the solubility of themutant proteins of the invention is improved if lysine residues arechemically altered by treatment with sulfo-N-hydroxysuccinimide-acetate(sulfo-NHS-acetate; which changes the positive charge of the epsilonamine group of lysine to a neutral charge),sulfo-succinimidyl-3-(4-hydroxyphenyl) propionate (which changes thepositive charge of the epsilon amine group of lysine to a neutral chargeand adds an aromatic functional group), or succinic anhydride (whichchanges the positive charge of the epsilon amine group of lysine to anegative charge). Therefore, one or more of the lysine residues of anunmutated CRP subunit or preCRP is preferably deleted or replaced withanother amino acid to improve the solubility of the resultant mutantprotein. Other suitable amino acid changes to increase the solubility ofthe mutant proteins of the invention include deleting one or morehydrophobic amino acids, replacing one or more hydrophobic amino acidswith charged amino acids, adding one or more charged amino acids, orcombinations of these changes. However, for the reasons stated above,the addition of lysine residues should be avoided. Aqueous media includewater, saline, buffers, culture media, and body fluids.

The mutant proteins of the invention can be prepared by expression ofDNA coding for them in transformed host cells. DNA coding for a mutantprotein according to the invention can be prepared by in vitromutagenesis of a CRP genomic or cDNA clone or can be chemicallysynthesized.

As discussed above, genomic and cDNA clones coding for human, mouse, andrabbit CRP have been isolated, and there is substantial homology betweenthe amino acid sequences of CRP's from different species. Given thesubstantial homology between CRP's from different species, probes canreadily be prepared so that genomic and cDNA clones can be isolatedwhich code for CRP's from other species. Methods of preparing suchprobes and isolating genomic and cDNA clones are well known [See, e.g.,Lei et al., J. Biol. Chem., 260, 13377-83 (1985); Woo et al., J. Biol.Chem., 260:13384-88 (1985); Hu et al., Biochem., 25:7834-39 (1986); Huet al., J. Biol. Chem., 263:1500-1504 (1988); Whitehead et al., Biochem.J., 266:283-90 (1990)].

Using one of the known clones or a newly-isolated clone, DNA coding fora mutant protein according to the invention can be prepared usingconventional and well known in vitro mutagenesis techniques.Particularly preferred is site-directed mutagenesis using polymerasechain reaction (PCR) amplification. See Example 2. The followingreferences describe other site-directed mutagenesis techniques which canbe used to produce DNA coding for a mutant protein of the invention:Current Protocols In Molecular Biology, Chapter 8, (Ansubel ed. 1987);Smith & Gilliam, Genetic Engineering Principles And Methods, 3:1-32(1981); Zoller & Smith, Nucleic Acids Res., 10:6487-6500 (1982); Zolleret al., Methods Enzymol., 100:468-500 (1983); Zoller & Smith, DNA,3:479-88 (1984); Brake et al., Proc. Natl. Acad. Sci. USA, 81:4642-46(1984); Bio/Technology, pages 636-39 (July 1984); Botstein et al.,Science, 229:1193 (1985); Kunkel et al., Methods Enzymol., 154:367-82(1987).

DNA coding for a mutant protein of the invention can also be prepared bychemical synthesis. Methods of chemically synthesizing DNA having aspecific sequence are well-known in the art. Such procedures include thephosphoramidite method [See, e.g., Beaucage and Caruthers, TetrahedronLetters, 22:1859 (1981); Matteucci and Caruthers, Tetrahedron Letters,21:719 (1980); and Matteucci and Caruthers, J. Amer. Chem. Soc.,103:3185 (1981)] and the phosphotriester approach [See, e.g., Ito etal., Nucleic Acids Res., 10:1755-69 (1982)].

To show that the mutant protein expresses neo-CRP antigenicity, it isusually sufficient to establish that the protein 1) reacts positivelywith an antibody specific for an epitope found only on mCRP or 2) bindsaggregated immunoglobulin (e.g., aggregated IgG).

The methods of the invention are described in further detail below, andrefer to the use of mCRP. It is contemplated that the mutant protein(s)disclosed above may be similarly employed.

In accordance with the method of the invention of stimulatingthrombocytopoiesis in a mammal, an effective amount of mCRP isadministered to the mammal. The term "thrombocytopoiesis" refersgenerally to the process by which thrombocytes, or platelets, are made.The mCRP may be administered to the mammal when a decrease inthrombocyte number, volume and/or mass in the mammal is first detected.The mCRP may also be administered at the time of, or after,administering to the mammal therapy which may adversely affectthrombocyte number, volume and/or mass. For instance, the mCRP may beadministered to the mammal within several hours of receiving relativelyhigh doses of X-irradiation. Alternatively, the mCRP may be administeredprophylactically, i.e., prior to therapy, to avoid decreased thrombocytenumber, volume, and/or mass. As an example, the mCRP is administered tothe mammal 1 to 5 days before the mammal is infused with a chemotherapydrug.

The mCRP may be administered to the mammal in apharmaceutically-acceptable carrier. Pharmaceutically-acceptablecarriers are well known to persons skilled in the art. For instance,suitable carriers for administering mCRP include fluids such as water,saline, and buffer. More preferably, a Tris or phosphate buffered salineis used as the carrier. It will be apparent to those persons skilled inthe art that certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of protein beingadministered.

The mCRP is preferably administered to the mammal by injection (e.g.,intravenous, intraperitoneal, subcutaneous, intramuscular). Effectivedosages and schedules for administering mCRP may be determinedempirically, and making such determinations is within the skill of theart. A dose of about 0.1 to 10 mg of mCRP per kilogram of body weight ofthe mammal will be effective for stimulating thrombocytopoiesis. A doseof about 3 mg of mCRP per kilogram of body weight is preferred. It isunderstood by those skilled in the art that the dose of mCRP that mustbe administered will vary depending on, for example, the mammal whichwill receive the mCRP, the nature of the medical condition or therapybelieved to be responsible for decreased thrombocyte numbers, volumeand/or mass, the extent of damage to the blood cell producing tissues,the route of administration, and the identity of any other drugs beingadministered to the mammal. It is also understood that it may benecessary to give more than one dose of mCRP. Generally, multiple dosesof mCRP must be given to the mammal. The interval between doses ispreferably from about 1 day to about 7 days. Administration of mCRPshould be continued until acceptable thrombocyte number, volume and/ormass has been restored to the mammal.

The invention also provides a method of treating thrombocytopenia in amammal. The term "thrombocytopenia" in the present invention is used ina broad sense and refers to the physiological condition in mammalsusually characterized by an abnormally low thrombocyte number in theperipheral blood.

In the method, the mammal is first diagnosed as suffering fromthrombocytopenia. Making the diagnosis is within the skill in the art.Those skilled in the art will also appreciate that different thrombocytelevels may warrant a thrombocytopenia diagnosis for different mammalianspecies. The diagnosis is usually made in humans when thrombocyte levelsin the peripheral blood fall below 100,000 cells per cubic millimeter.Thrombocytopenia can be the result of a disorder of production,distribution or destruction of thrombocytes or thrombocyte-producingcells. To treat the thrombocytopenia, the mCRP is administered to themammal according to the modes and schedules of administration describedabove.

The thrombocytopoietic activity in the mammal can be measured ormonitored in various ways. For instance, the activity can be measuredusing in vitro assays. One such assay is the colony forming assay. Theseassays are known in the art and generally involve harvesting bone marrowcells from the mammal and culturing the cells in a clot or soft agar[Mizoguchi et al., Exp. Hematol., 7:346 (1979); Metcalf, D., InHematopoietic Colony Stimulating Factors, Elsevier/North Holland,Amersterdam (1984)]. By using select reagents and stains in the colonyforming assays, the following activities can be measured or monitored:(1) basic formation and growth of megakaryocyte colonies (Meg-CSFactivity) and (2) potentiation/maturation of megakaryocytes during laterphases leading up to and during platelet release (Meg-POT activity). Inaddition, there are in vitro liquid culture assays. For example,acetylcholinesterase (AChE) enzyme activity quantitatively correlateswith megakaryocyte numbers in liquid culture [See, Burstein et al., J.Cellular Phys., 122:159 (1985)]. Those skilled in the art willappreciate, however, that the AChE assay may not be suitable in someinstances since AChE is not found in all mammalian megakaryocytes.

The thrombocytopoietic activity in the mammal can also be monitored byperipheral blood or bone marrow analysis. Thrombocyte levels in thecirculating blood can be determined by cell count analysis. The cellcount analysis may be performed by measuring a Coulter Panel using theCoulter Model S-plus instrument. Thrombocytopoietic activity may also beassessed by examining cells morphologically. For example, bone marrowsamples can be obtained from the mammal and prepared for microscopyusing standard histological techniques known in the art. By staining thebone marrow cells, one can observe the size and number of megakaryocytesin the marrow sample.

Although not fully understood, it is believed that the stimulatingeffect of mCRP on thrombocytopoiesis in vivo may be due, at least inpart, to the localization of mCRP to the bone marrow. Data (not shown)has indicated that intravenously-injected mCRP rapidly localized to thebone marrow in mice within 4 hours after administration and remainedthere for at least 24 hours.

The mCRP can also be employed to stimulate megakaryocytopoiesis andthrombocytopoiesis in vitro. In a preferred embodiment, there isprovided a method for promoting megakaryocyte growth and maturation invitro. The method comprises the steps of providing hematopoietic cellsin a cell culture medium and culturing the cells in a culture vessel inthe presence of an effective amount of mCRP. The use of mCRP to promotemegakaryocyte growth and maturation in vitro has a variety ofapplications. For instance, the mCRP can be used in bone marrow colonyforming assays to screen and detect the extent of cell damage caused byradiation or drug therapy in vivo. It is also believed thathematopoietic cells grown in vitro in the presence of mCRP can beinfused back into hematopoietically compromised patients.

In the method, hematopoietic cells are provided in a cell culturemedium. The term "hematopoietic cells" is used to refer to cells or cellsamples suspected of containing pluripotent or immature stem cells(which may be megakaryocyte precursor cells) or cells of themegakaryocytic lineage. Although the preferred method utilizes bonemarrow cells, it is contemplated that other hematopoietic cell samplessuspected of containing stem cells or cells of the megakaryocyte lineagemay be employed. The hematopoietic cell samples may be obtained, forexample, from peripheral blood or from umbilical cord blood.

The cells used in the method may be obtained directly from the mammal.For instance, bone marrow cells can be obtained by flushing the femurbones of the mammal. The cells can also be obtained through varioustechniques known in the art, including but not limited to, needle biopsyor needle aspiration. Preferably, the cells are obtained underrelatively aseptic or sterile conditions.

The cells are then placed in an aqueous isotonic or buffered medium.Preferably, the cells are placed in a sterile tissue culture medium.Suitable tissue culture media are well known to persons skilled in theart and include, but are not limited to, Basal Medium Eagle ("BME"),Minimal Essential Medium ("MEM"), RPMI-1640, Dulbecco's Modified Eagle'sMedium ("DMEM"), and McCoy's 5A Medium. These tissue culture media maybe purchased commercially from various sources, including Sigma ChemicalCompany (St. Louis, Mo.) and GIBCO (Grand Island, N.Y.).

More preferably, the cells are placed in a sterile tissue culture mediumcontaining a sufficient amount of antibiotic agent to prevent microbialcontamination. Penicillin, streptomycin, and gentamicin, as well asother tissue culture grade antibiotics known in the art, arecommercially available from Sigma and GIBCO, and may be added to theaqueous medium in concentrations recommended by the manufacturer.Microbial contamination may also be reduced by the use of routinesterile techniques in handling the cells and the use of a laminar flowhood.

The cells are then preferably counted to determine the approximateconcentration and viability of the cells. Concentration and viability ofthe cells may be determined by standard techniques known in the art. Forexample, cell viability may be determined by trypan blue exclusionmethods.

The cells are then cultured in a suitable cell culture medium underconditions sufficient for the cells to remain viable and grow.Preferably, the cell culture medium comprises a sterile tissue culturemedium supplemented with a sufficient quantity of nutrient components.Suitable tissue culture media include, but are not limited to, BME, MEM,RPMI-1640, DMEM, and McCoy's 5A medium, all of which are commerciallyavailable from Sigma or Gibco. Nutrient components contemplated by theinvention include, but are not limited to, amino acids, lipids andhormones. The cell culture medium also preferably includes at least oneantibiotic agent to prevent microbial contamination.

The cells can be cultured in a variety of ways, including culturing in aclot, in agar, or in liquid culture [Mizoguchi et al., Exp. Hematol.,7:346 (1979); Metcalf, D., In Hematopoietic Colony Stimulating Factors,Elsevier/ North Holland, Amersterdam (1984); Burstein et al., J.Cellular Phys., 122:159 (1985)]. The cells are preferably cultured in avessel suitable for sterile tissue culture, including but not limitedto, plates and dishes. The culture vessel may be formed from a varietyof materials such as glass or plastic. Culture vessels are commerciallyavailable from COSTAR (Cambridge, Mass.) and Corning (Corning, N.Y.) Theoptimum number of cells cultured in the culture vessel may be determinedempirically by those persons skilled in the art without undueexperimentation. In a preferred embodiment, about 5×10⁵ bone marrowcells/ml are cultured in a Petrie dish.

In one embodiment, bone marrow cells are cultured in agar. For culturingpurposes, both the culture vessel and agar should be sterile. Agarsolutions may be sterilized by heating to relatively high temperatures.Preferably, the agar solution is sterilized by autoclaving for 15minutes (250° F. at 15 psi). Upon heating, the agar will go intosuspension. Upon cooling, the solution will become a semi-solid gel. Inanother embodiment, bone marrow cells are cultured in a plasma clotattached to a culture vessel. An example of this culturing method isfurther described in Example 3 below. In a further embodiment, the bonemarrow cells are cultured in a continuous liquid culture.

The cells are cultured in the culture vessel in the presence of aneffective amount of mCRP. The concentration of mCRP in the reagent mayvary and will be added to the culture at a dose determined empiricallyby those in the art. The concentration of mCRP in the culture willdepend on various factors, such as the kind of cell culture mediumemployed, and the length of time of the cell culturing period. Thedesired concentrations may be determined empirically and it is withinthe skill in the art to make such determinations. As described inExample 3, Applicant found that about 2 to 20 microgram/ml mCRP waseffective for promoting megakaryocyte colony formation and maturation invitro. For comparative purposes, appropriate controls should also becultured and tested to determine the efficacy of the mCRP and toquantitate results.

The culture vessels containing the cells and mCRP (or controls) are thenincubated under conditions sufficient for the cells to remain viable andgrow. Preferably, the culture vessels are incubated in a humidifiedchamber for about 4 to about 7 days. The specific temperature and timeof incubation, as well as other culture conditions, can be varieddepending on such factors as the concentration of the mCRP. Thoseskilled in the art will be able to determine operative and optimalculture conditions without undue experimentation. Growth and maturationof megakaryocytes in the cultures can then be determined by counting thenumber of colonies formed in each culture vessel and/or by examining themorphological characteristics of the cells under a microscope.

The invention further provides an article of manufacture and kitcontaining materials useful for stimulating thrombocytopoiesis andmegakaryocytopoiesis. The article of manufacture comprises a containerwith a label. Suitable containers include, for example, bottles, vials,and test tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for stimulating thrombocytopoiesis and megakaryocytopoiesis.The active agent in the composition is mCRP or mutant protein havingneo-CRP antigenicity. The label on the container indicates that thecomposition is used for stimulating thrombocytopoiesis andmegakaryocytopoiesis, and may also indicate directions for either invivo or in vitro use, such as those described above.

The kit of the invention comprises the container described above and mayfurther include other materials desirable from a commercial and userstandpoint, including but not limited to buffers, diluents, filters,needles, syringes, cell culture medium and culture vessels.

EXAMPLES Example 1 Isolation and Purification of mCRP A. CRP Preparationand Purification

Native CRP was isolated from pleural or ascites fluid bycalcium-dependent affinity chromatography usingphosphorylcholine-substituted BioGel® A 0.5 m (an agarose-based resinobtained from BioRad Laboratories) as described by Volanakis et al. [J.Immunol., 113:9-17 (1978)] and modified by Potempa et al., [Mol.Immunol., 24:531-41 (1987)]. Briefly, the pleural or ascites fluid waspassed over the phosphorylcholine-substituted column, and the CRP wasallowed to bind. Then, the column was exhaustively washed with 75 mMTris-HCl-buffered saline (pH 7.2) containing 2 mM CaCl₂ until theabsorbance at 280 nm was less than 0.02. The CRP was eluted with 75 mMTris, 7.5 mM citrate-buffered saline (pH 7.2). This high concentrationof Tris significantly reduces non-specifically adsorbed proteins whichoften contaminate affinity-purified CRP preparations. CRP-containingfractions were pooled, diluted three-to-five fold with deionized water,adsorbed to Q-Sepharose Fast Flow® ion exchange resin, and then elutedwith a linear salt gradient from 0-1M NaCl in 10 mM Tris-HCl, pH 7.4.CRP-containing fractions were pooled and re-calcified to 2-5mM CaCl₂ (byadding a suitable amount of a 1M solution) and applied to unsubstitutedBiogel® A 0.5 m column to remove residual serum amyloid P component("SAP"). Then, the CRP was concentrated to 1 mg/ml using ultrafiltration(Amicon; PM30 membrane) under 10-20 psi nitrogen. A CRP extinctioncoefficient (mg/ml) of 1.95 was used to determine concentration. Next,the concentrated CRP was exhaustively dialyzed against 10 mMTris-HCl-buffered saline, pH 7.2, containing 2 mM CaCl₂. Thispreparation produced a single Mr 23,000 band on SDS-PAGE electrophoresisand was more than 99% free of SAP, IgG and all other proteins tested forantigenically.

For the Q-Sepharose Fast Flow® column, 5 ml of the final concentratedCRP solution containing 5 mg of purified native CRP was diluted in about30 ml of 10 mM Tris-HCl, pH 7.4, and the resulting solution was loadedon the Q-Sepharose Fast Flow column at 6 ml per minute. Bound materialwas eluted with a linear NaCl gradient using 10 mM Tris-HCl, pH 7.4,containing 1M NaCl. A₂₈₀ was measured using the BioPilot® automatedchromatography system.

B. mCRP Preparation

To make mCRP, purified native CRP, prepared as described above, at 1mg/ml was incubated in 8M ultra-pure urea in the presence of 10 mM EDTAfor one hour at 37° C. The urea was removed by dialysis into 10 mMsodium phosphate buffer (pH 7.4) or Tris-HCl buffer (pH 7.2) containing0.015M sodium chloride. The mCRP was sterile filtered through a 0.2micron filter (Gelman). Some of the sterile filtered mCRP was adjustedto physiologic ionic strength by adding sodium chloride to give a finalconcentration of 0.15M NaCl, and then incubated in an ice bath for 15minutes. Before adjusting the ionic strength, the mCRP is soluble. Afteradjusting the ionic strength, the majority of mCRP self-aggregates intoa suspension.

Example 2 Preparation of a Mutant Subunit of mCRP

This example describes the preparation of a recombinant DNA moleculecoding for a mutant human CRP subunit in which the two cysteine residuesat positions 36 and 97 (using the numbering system of Woo, et al., J.Biol. Chem., 260, 13384-13388 (1985)) have been replaced with alanineresidues. This example also describes the preparation of vectorscontaining the recombinant DNA molecule operatively linked to expressioncontrol sequences and the expression of the mutant CRP subunits inEscherichia coli.

A. Replacement Of The Codons For Cysteine-36 And Cysteine-97 In The CRPCoding Sequence Using A Polymerase Chain Reaction Technique

To replace codons for cysteine-36 and cysteine-97 in the coding sequencefor mature human CRP subunits with codons for alanine, the method ofHorton et al., BioTechnicues, 8, 528-535 (1990) which is based on thepolymerase chain reaction (PCR; Saiki et al., Science, 239, 487-491(1988)) was used. Since two independent changes were desired, theprocess was modified to incorporate a total of five PCR reactions asillustrated in FIG. 1A. Table 1 shows the sequence of theoligonucleotides used as primers in the PCR reactions.

                  TABLE 1                                                         ______________________________________                                        Num-                                                                            ber Sequence SEQ ID NO                                                      1    5'GGGCCATATGCAGACAGACATGTCGAGG → 3'                                                              1                                                         NdeI                                                                  - 2 3' ← TCGGAAGTGACACCGCGAAG5'    2                                    3           5'CACTGTGGCGCTCCAC → 3'  3                                                    HhaI                                                        - 4 3' ← GGTCATGTGTAGCGCTGTT5'    4                                     5           5'CACATCGCGACAAGCTG → 3'  5                                                  NruI                                                         - 6 3' ← GGACTTCCATGGAGTCTAGAGCGG5'   6                                               KpnI    BglII                                                 ______________________________________                                         Italicized bases indicate restriction enzyme recognition sites.               Bold bases indicate mutagenized codons and the initiator ATG codon.           Underlined bases are complementary to the CRP cDNA sequence.             

As shown in FIG. 1A, the five reactions were: (1) Reaction of cDNA clonecoding for preCRP with primers 1 and 2 to produce PCR product A; (2)Reaction of cDNA coding for preCRP with primers 3 and 4 to produce PCRproduct B; (3) Reaction of cDNA coding for preCRP with primers 5 and 6to produce PCR product C; (4) Reaction of products A and B in thepresence of primers 1 and 4 to produce PCR product D1; and (5) Reactionof products D1 and C in the presence of primers 1 and 6 to produce thefinal product D2. D2 was thought to code for the mature sequence ofhuman CRP subunit (the presequence has been eliminated), except thatthere was an additional methionine at the N-terminus and cysteines 36and 97 had been replaced by alanines. This was subsequently found not tobe the case (see section F below).

The DNA coding for human preCRP used as the starting material for thesePCR reactions was obtained by digestion of pCRP5 with EcoRI to yieldlinear (noncircular) DNA. A sample of plasmid pCRP5 was obtained fromDrs. Bruce Dowton and Harvey Colten of Washington University School ofMedicine, St. Louis, Mo. pCRP5 was isolated from a human liver cDNAlibrary as described in Tucci et al., J. Immunol., 131, 2416-19 (1983).The nucleotide sequence of the cDNA of pCRP5 and the amino acid sequenceof the preCRP coded for by it are given in Woo, et al., J. Biol. Chem.,260, 13384-13388 (1985). However, as described in detail in section Fbelow, the cDNA of the pCRP5 sample obtained from Drs. Dowton and Coltenwas found to have a deletion in codon 47.

The PCR reactions were carried out using VENT polymerase (New EnglandBiolabs) to minimize unwanted mutations due to misincorporation ofbases, and the PCR reactions were done using 20 cycles, each cycleconsisting of: 94° C. for 1 minute; 37° C., 42° C. or 60° C. for 1minute (the annealing temperature depending on the sequence of theprimers); and 74° C. for 3 minutes. Following the amplification steps,the reactants were further incubated at 74° C. for 5 minutes to completethe synthesis of double-stranded DNA. Each of the products was purifiedby agarose gel electrophoresis as described in Horton et al., supra. Forthe PCR reactions where the template consisted of two overlappingsequences (PCR D1 and PCR D2 in FIG. 1A), the reactants were incubatedwithout primers for 4 cycles to allow the formation of full-lengthtemplate before normal amplification was carried out.

PCR products were digested with restriction endonucleases HhaI and NruI(see Table 1) to confirm that the products incorporated the desiredmutations.

B. Construction Of A Plasmid For The Overexpression Of The Mutant CRPSubunit

The final PCR product D2 was concentrated by filtration through aCentricon 30 apparatus (Amicon, Beverly, Mass.), and then treated withT4 polynucleotide kinase (Pharmacia, Piscataway, N.J.) and T4 DNA ligase(New England Biolabs, Inc.) as described in Denney et al.,Amplifications, 4, 25-26 (1990). The resultant material was digestedwith NdeI and BglII to release the mutant CRP coding sequence, and thereleased coding sequence was ligated to the expression vector pETV whichhad been digested with NdeI and BamHI and treated with calf intestinalalkaline phosphatase (Promega, Madison, Wis.). The ligation mixture wasused to transform E. coli DH5α (Gibco BRL Life Technologies, Inc.), andtransformants were screened by minipreps performed as described inBirnboim et al., Nucleic Acids Res., 7, 1513-1523 (1979) to identify thecorrect plasmid pIT4. A restriction map of pIT4 is provided in FIG. 1B.As shown in FIG. 1B, the mutant CRP coding sequence is under the controlof the T7 promoter.

Plasmid pETV is a derivative of pET3a (see FIG. 2A). The preparation ofpET3a is described in Rosenberg et al., Gene, 56, 125-135 (1987).Plasmid pET3a was obtained from Dr. W. Studier, Brookhaven NationalLaboratory, Upton N.Y. Plasmid pET3a has two NheI restriction enzymesites (see FIG. 2B). One is located at the T7 gene 10 translation startsite in which it was desired to insert the mutant CRP coding sequence.The second NheI site is located within a 190-bp fragment bounded byEcoRV sites. This second NheI site was eliminated by digestion withEcoRV and recircularizing the plasmid to yield pETV.

The predicted sequence of the junction between the expression system ofpETV and the coding region for mutant CRP subunit in pIT4 was confirmedby DNA sequencing as follows. Plasmid pIT4 was digested with XbaI andKpnI, and the relevant fragment subcloned into M13mp18RF (Yanisch-Perronet al., Gene, 33, 103-119 (1985)). Single-stranded DNA was obtained fromcultures carrying M13mp18RF and sequenced as described by Sanger et al.,J. Mol. Biol., 94, 441-558 (1975) using an Applied Biosystems Model 370AAutomated DNA Sequencer.

C. Expression And Purification Of The Mutant CRP Subunit Produced BypIT4

Plasmid pIT4 was used to transform E. coli BL21(DE3) (preparationdescribed in Studier et al., J. Mol. Biol., 189, 113-130 (1986)) whichcarries the phage T7 RNA polymerase gene under expression control of thelacUV5 operator and promoter. Competent E. coli BL21(DE3) was obtainedfrom Novogen, Madison, Wis. Transformants were selected on LB medium(Miller, Experiments In Molecular Genetics (1972)) containing 50 μg/mlampicillin.

Transformed cells were grown in M9ZB medium (Studier et al., J. Mol.Biol., 189, 113-130 (1986)) containing 100 μg/ml ampicillin at 37° C.for small-scale experiments. Ten-liter cultures were grown in a NewBrunswick Microgen SF-116 fermentor in 2YT medium (Miller, ExperimentsIn Molecular Genetics (1972)) plus 0.4% (w/v) glucose and 100g/mlampicillin at 37° C. with aeration using compressed air at a rate of 10liters per minute.

Synthesis of the T7 RNA polymerase, and consequently of the mutant CRPsubunit, was induced with 1 mM (final concentration)isopropyl-beta-D-thio-galactoside (IPTG; Boehringer Mannheim) when thecell density reached OD₆₀₀ =4. The cells were harvested 3 hours afterinduction by rapidly mixing the culture with an equal volume of ice,concentrating the cells by filtration using a Millipore Pelliconapparatus equipped with a Durapore 0.5 μM membrane, and centrifuging thecells in a Beckman JA10 rotor at 10,000 rpm for 20 min.

The harvested cells were suspended in 20mM Tris-HCl, pH 7.5, containing5mM EDTA (40 g in 500 ml) and disrupted by three passages through aManton-Gaulin homogenizer at 10,000 psi. The extract was centrifuged ina Beckman JALO rotor at 8,000 rpm for 20 min. at 2° C. The pelletcontaining the insoluble mutant CRP subunits was washed twice with 20 mlof the same buffer containing 0.5% (v/v) Triton X-100 (Bio-Rad),followed by centrifugation in a JA18 rotor at 9,000 rpm for 20 min. toremove the soluble material.

About 50-100 mg of the final pellet was dissolved in 6M Guanidinium-HCl(J. T. Baker, Phillipsburg, N.J.) in 15 mM Tris-HCl, 10 mM EDTA buffer(pH 7.2). Insoluble materials were removed by centrifugation anddiscarded. Soluble materials were filtered through 0.8/0.2 micronfilters (Gelman) and were made 28% ammonium sulfate by adding theappropriate percentage of saturated (100%) ammonium sulfate solution(Sigma Chemical Co., St. Louis, Mo.). Pellet was collected bycentrifugation and washed in Tris-buffered saline (pH 7.4). Pellet wasthen dissolved at 1.4 mg/ml in 8M urea, 25 mM acetate buffer (pH 4.0)and was loaded onto a S-Sepharose-FF® cation exchange column (Pharmacia)equilibrated with 6M urea, 25 mM acetate buffer (pH 4.0). Loosely boundcontaminating materials were removed by washing the column with 6M urea,25 mM acetate buffer (pH 4.0) containing 0.5M sodium chloride. Thecolumn was reequilibrated with the above buffer lacking sodium chloride.Bound mutant recombinant-CRP ("mutant rCRP") was eluted using 6MGuanidinium-HCl in 25 mM acetate buffer (pH 4.0). Absorbance at 280 nmwas measured using a BioPilot automated chromatography system(Pharmacia), and an elution profile obtained.

Eluted mutant rCRP was immediately applied to a Superdex 200® molecularsieve column (Pharmacia) equilibrated in 4M urea, 3M Guanidinium-HCl, 25mM acetate buffer (pH 4.0). Protein was passed through this column at arate of 30.5 cm/hr. Chromatographed proteins were separated andcollected based on absorbance at 280 nm using the automated BioPilot®Chromatography System (Pharmacia). Protein fractions containing mutantrCRP were sterile filtered through 0.8/0.2 micron filters (Gelman),pooled, and dialyzed into 25 mM Tris-HCl, 0.015M NaCl (pH 7.4).

D. Characterization Of The Fractions Eluted From The Superdex 200®

The peaks from the Superdex 200® column were analyzed by dot blot,Western blot, SDS-PAGE and ELISA. Mutant rCRP inclusion bodypreparations were sometimes also tested. Native CRP and mCRP weresometimes used as controls. The preparation of all of these materials isdescribed in the previous section.

1. Dot Blot Assays

Following a modification of the procedure of Zhang, J. Immunol., 138:575(1987), nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.)were pre-soaked in TBS (25 mM Tris-HCl, 0.15M NaCl, pH 7.4) for 30 min.,and excess buffer was removed with filter paper. The membranes were thenfitted into a Bio-Dot® microfiltration apparatus (Bio-Rad). Aliquots (50μl) of the various test proteins at 5 μg/ml were dotted onto themembrane, incubated overnight at 4° C., and then vacuum-filtered toremove all of the liquid from the wells. Blocking solution (100 μl of itBSA in TBS) was added to the wells and incubated for 30 min. at roomtemperature (RT), and vacuum-filtered through the membrane. The wellswere washed three times with TBS containing it BSA and 0.05% Tween 20(TBS washing buffer). Mouse monoclonal antibody 3H12 or other class IIIor class IV monoclonal antibodies (U.S. Pat. No. 5,272,257) and goatpolyclonal anti-neo-CRP antiserum were added and incubated for 30 min.at RT followed by washing. Monoclonal antibody specific for mCRP wasused unlabeled, and the blots were developed by adding rabbit anti-mouseIgG F(ab')₂ labeled with horseradish peroxidase (Southern BiotechnologyAssociates, Birmingham, Ala.), incubating for 30 min. at RT, addingperoxidase substrate 4-chloro-2-naphthol (Bio-Rad) in 10 mM Tris-HCl,0.15M NaCl, containing methanol and H₂ O₂ prepared as directed(Bio-Rad), and incubating for 30 min. at RT to allow for colordevelopment. Polyclonal anti-neo-CRP was labeled with horseradishperoxidase, and the blots were developed by adding 4-chloro-2-naphtholfollowed by an incubation for 30 min. at RT for color development.

Monoclonal antibody 3H12 is an IgG antibody specific for an antigenicdeterminant found on mCRP but not on native CRP. Its preparation andproperties are described in U.S. Pat. No. 5,272,257 and in Ying et al.,J. Immunol., 143:221-228 (1989). Anti-neo-CRP polyclonal antiserum is anantiserum prepared by immunizing a goat with mCRP in complete Freund'sadjuvant and then affinity-purifying the harvested antiserum by passingit over a column substituted with strongly bound mCRP. The resultingaffinity-purified anti-neo-CRP antiserum was predominantly reactive forthe neo-CRP antigenicity expressed by mCRP but not by native CRP.Polyclonal anti-neo-CRP was labelled with horseradish peroxidase asdescribed in Potempa et al., Mol. Immunol., 24:531-541 (1987).

The dot blots showed that mCRP and mutant rCRP reacted with monoclonalantibody 3H12 and polyclonal anti-neo-CRP-HRP, indicating that all ofthese materials express antigenic determinants found on mCRP but not onnative CRP. Native CRP did not react with antibodies 3H12 andanti-neo-CRP-HRP as expected.

2. Western Blot

The peaks were also analyzed by Western blot. To perform the Westernblot, 5-10 μl of the peak concentrates were electrophoresed on 12%SDS-PAGE gels under reducing and non-reducing conditions. Afterelectrophoresis, protein was transferred to a nitrocellulose membraneusing the JKA Biotech (Denmark) Semidry Electroblotter. The remainder ofthe procedure was the same as described above for the dot blot, exceptthat three mouse mAbs (3H12, 2C10 and 8C10) were used. The color wasdeveloped as described in the previous section for mAb 3H12.

Monoclonal antibody 3H12 is described above. Monoclonal antibody 8C10reacts with a determinant found only on mCRP, whereas mAb 2C10 reactswith a determinant found on both native CRP and mCRP. The preparationand properties of mAbs 2C10 and 8C10 are described in U.S. Pat. No.5,272,257; Ying et al., J. Immunol., 143:221-228 (1989); Ying et al.,Mol. Immunol., 29:677 (1992).

The Western blot results showed that mCRP and mutant rCRP reacted withall three mAbs, indicating that all of these materials express antigenicdeterminants found on mCRP. Native CRP reacted with mAb 2C10, but notwith mAbs 3H12 and 8C10, confirming that this material is native CRP andthat the antibodies were reacting as expected.

The Western blot results also showed that the predominant materialpresent in mutant rCRP (the major peak obtained when the mutant CRPsubunit was chromatographed on Superdex 200®) was free, monomericsubunits. Thus, mutant rCRP produced by replacing the cysteine residuesin a CRP subunit, was processed to give a pure, well-defined product.

3. SDS-PAGE

The peak concentrates were run on PhastGel® SDS-PAGE gels (Pharmacia). Agradient of 8-25% gradient acrylamide was used. After theelectrophoresis was complete, the gels were stained with Coomassie blue.The results are shown in FIG. 3.

Lane 1 contains the mutant rCRP inclusion body preparation. Note thatthere are two predominant bands, including one at approximately the sameposition as the single mCRP band (Mr of about 23,000). This band wasverified by Western blot analysis to be antigenically reactive withantibodies specific for mCRP determinants. The second predominant bandof Mr about 7000 was verified by Western blot to also be antigenicallyreactive with antibodies specific for mCRP determinants.

Lanes 2 and 16 contain washed inclusion bodies. Lane 3 contains thesodium chloride wash from the S-Sepharose FF® cation exchange column.Lanes 4 and 5 contain the Guanidinium-HCl eluate from the cationexchange column. Note that only one major band was obtained having amolecular weight of approximately 23,000, the molecular weight of thedesired free mutant CRP subunits. As can be seen, there are many fewerbands as compared to the mutant rCRP inclusion body preparation (lanes 1and 2), and the amounts of remaining contaminants, especially the onepredominant contaminant having Mr of approximately 7000 have beensubstantially reduced. Thus, the purity of the mutant CRP subunit wasgreatly improved by the single-step S-Sepharose-FF® chromatographyprocedure.

Lanes 6 and 15 contain molecular weight standard protein bands.

Lanes 7 and 8 contain large molecular weight aggregate proteinsseparated from mutant rCRP-cation exchange protein by Superdex 200® gelfiltration chromatography. Note that some protein of Mr 23,000 isrecovered in these fractions. These proteins are discarded.

Lanes 9, 10 and 11 contain highly-purified mutant rCRP. By Western blotanalysis, this band reacted with antibodies specific for mCRP. No otherbands of protein were visualized in these samples using the Western blottechnique. Lane 12 contains a fraction of mutant rCRP contaminated withsmaller Mr proteins. Such fractions, even though they containsubstantial amounts of mutant rCRP, are discarded. Lane 13 containssmall contaminating proteins which are discarded. Lane 14 contains smallmolecular weight fractions of mutant rCRP samples chromatographed onSuperdex 200®. This sample reacted with antibodies specific for mCRP byWestern blot. Thus, these proteins contain a fragment of mutant rCRPmolecule.

4. ELISA

Finally, an ELISA was performed to detect binding of antibodies specificfor native CRP and mCRP to the materials in the peaks eluted from theSuperdex 200® column. A direct binding ELISA was used for mCRP andmutant rCRP preparations. A ligand capture ELISA was used for native CRPpreparations.

In the direct ELISA, 100 μl of each test protein (5 μg/ml) in 50 mMsodium bicarbonate buffer (pH 9.5) were placed in the wells of Nuncpolystyrene plates (Scientific Supply, Shiller Park, Ill.) and incubatedfor 2 hours at 37° C. or 4° C. overnight. The wells were blocked withTBS (25mM Tris-HCl, 0.15M NaCl, pH 7.4) containing 1% bovine serumalbumin (BSA) (TBS-A) for 60-120 min. at 37® C. The wells were washedwith TBS containing 0.05% Tween 20 (TBS-wash buffer). Antibodies wereserially diluted with TBS-A, and 100 μl aliquots were added to the wellsand incubated for 60 min. at 37° C., followed by washing.Peroxidase-conjugated rabbit anti-mouse IgG (Southern Biotech) in TBS-Awas added to the wells for 60 min. at 37° C. After washing, 100 μl ABTSsubstrate (2-2'azino-bis 3-ethylbenzylthiazoline-6-sulfonic acid, SigmaChemical Co.) were added per well and incubated for about 5-15 min. atRT. Plates were read at an absorbance of 414 nm on a Titertek® multiskanplate reader (Flow Laboratories, Helsinki, Finland).

For the ligand capture ELISA, plates were incubated with 100 μl/well ofPC-KLH (5 μg/ml) in bicarbonate buffer for 2 hr. at 37° C. or overnightat 4° C. Wells were blocked with TBS-A containing 2 mM CaCl₂ asdescribed above. After blocking, 100 μl/well of native CRP (5 μg/ml) inTBS-A containing 2 mM CaCl₂ were added and incubated for 60 min. at 37°C. After washing, the rest of the assay was performed as describedabove, except that 2 mM CaCl₂ was included in all buffers. PC-KLH isphosphorylcholine (PC) substituted Keyhole Limpet hemocyanin (KLH). Itwas prepared by incubating KLH (Sigma Chemical Co.) with paranitrophenylphosphorylcholine (Sigma Chemical Co.) diazotized as described byChesebro and Metzger, Biochemistry, 11:766 (1972). The finalderivatization resulted in 28-52 moles PC per Mr of 1×10⁵ KLH.

The results of these ELISAs are shown in FIGS. 4A-C. FIG. 4A shows thereactivity of mCRP, mutant rCRP and native CRP with mAb 1D6. As expectedmAb 1D6 reacted with native CRP but not with mCRP. Also, mutant rCRPreacted like mCRP and unlike native CRP, indicating that the native CRPepitope recognized by mAb 1D6 is not expressed on mutant rCRP.

FIG. 4B shows the reactivity of mCRP and mutant rCRP with mAb 3H12.Monoclonal antibody 3H12 is specific for an antigenic determinant foundon the C-terminal octapeptide of mCRP. As expected mAb 3H12 reacted withmCRP. As shown, mutant rCRP, like mCRP, reacted with mAb 3H12,indicating that the neo-CRP epitope recognized by mAb 3H12 is alsoexpressed on mutant rCRP.

FIG. 4C shows the reactivity of mCRP and mutant rCRP with goatanti-neo-CRP-HRP polyclonal antiserum. As shown, mutant rCRP reactedwith goat anti-neo-CRP-HRP in a manner similar to mCRP. This suggeststhat the recombinant mutant protein produced by pIT4 is antigenicallyvery similar to mCRP.

F. Discovery Of A Deletion In Codon 47 And Preparation Of A Clone HavingThe Correct Sequence

Although mutant rCRP expression was obtained using E. coli BL21(DE3),the level of mutant rCRP produced was considerably lower than has beenreported in the literature for other proteins using the T7 RNApolymerase system employed in E. coli BL21(DE3). Possible reasons forthis low level of expression were explored, and it was noted that afragment of ˜7000 Mr was consistently observed as a product (see FIG.3). Western Blot analysis showed that this fragment was related to mCRPantigenically (see section D3 above).

N-terminal sequence analysis of the purified fragment confirmed that thefragment contained the expected CRP sequence from residues 1 to 6. TheN-terminal sequence analysis was performed by Analytical BiotechnologyServices (Boston, Mass.) using automated Edman degradation on an AppliedBiosystems Model 477A protein sequencer. The Mr 7000 fragment used forthe N-terminal sequencing was purified as follows. The concentration ofprotein in an inclusion body suspension was estimated using theBicinchoninic Acid protein assay (Pierce Biochemicals). Inclusion bodyprotein was then solubilized in 6M guanidine HCl (GuHCl; J. T. BakerInc.), 15 mM Tris-Cl, pH 7.2, 10 mM EDTA at a concentration of 4 mgprotein/ ml. Ammonium sulfate was added to 28% saturation, and theprecipitated protein collected by centrifugation (10 minutes at 12,000rpm in a Sorvall GSA rotor). The resulting pellet was washed with TBS(10 mM Tris-Cl, pH 7.4, containing 0.15M NaCl) and subsequentlydissolved in 8M urea, 25 mM sodium acetate, pH 4.0, at a concentrationof approximately 1 mg protein/ml as judged by absorbance at 280 nm. Thismaterial was passed over an S-Sepharose Fast Flow column (Pharmacia)equilibrated with 6M urea, 25 mM sodium acetate, pH 4.0. The column wasthen developed with a four-column volume gradient of 0-0.25M NaCl in theequilibration buffer (6M urea, 25 mM sodium acetate, pH 4.0). Thefragment eluted at approximately 0.24M NaCl. Fractions containing thefragment were pooled and concentrated by precipitation with 50%saturated ammonium sulfate. The precipitate was collected bycentrifugation as described above and dissolved in equilibration buffer.The dissolved precipitate was again passed over an S-Sepharose Fast Flowcolumn as described above, except that elution was performed with a0-0.8M NaCl gradient.

The size of the fragment and the quantity produced were very consistentfrom batch to batch. Furthermore, there was little evidence of otherfragments, suggesting that the 7000 Mr fragment resulted from incompletemRNA or protein synthesis rather than from proteolytic degradation.

Accordingly, the DNA coding for the mutant rCRP was sequenced. To do so,the coding sequence was excised from pIT4 using XbaI and KpnI andligated into pBluescript KS (Stratagene Inc.) previously digested withthe same enzymes. The subcloned DNA was sequenced on an AppliedBiosystems Sequenator by the dideoxy chain termination method usingstandard M13-based primers. The DNA sequences of two independentisolates revealed two differences with respect to the published Woo etal. sequence (Woo et al., J. Biol. Chem., 260, 13384-13388 (1985)).First, there was a synonymous substitution (G to A) in the wobbleposition of codon 152 (as noted above, the numbering system of Woo etal., J. Biol. Chem., 260, 13384-13388 (1985) is being used; this wouldactually be the 153rd codon of the CRP coding sequence of pIT4 since anATG start codon was added at the 3' end of the coding sequence (seesection A above). Second, there was a missing T at codon 47. The missingbase is in a GC-rich region, and it was initially thought that theabsence of this T from the sequence was due to compression, a commonsequencing artifact.

However, the results obtained from C-terminal sequence analysis of the7000 Mr fragment were consistent with the DNA sequence data. C-terminalsequencing of the purified fragment (prepared as described above) wasperformed by Analytical Biotechnology Services. Briefly, the fragmentwas digested with carboxypeptidase Y at a ratio of 5 μg enzyme per 10nmol protein (based on an estimated molecular weight of 7000). Themixture was incubated at 37° C., and 0.5 nmol aliquots were removed at0, 30, 60 and 180 minutes and frozen at °20° C. Each aliquot was thensubjected to amino acid analysis using the Waters PICO-TAG system. Thefollowing residues were released in roughly equal amounts afterdigestion of the fragment with carboxypeptidase Y: Ser, Tyr, Phe, Gly,Leu, Arg, and Ile. While these amino acids could possibly be assigned tothe CRP sequence ending at Phe 52:

    ______________________________________                                        Leu Ser Ser Thr Arg Gly Tyr Ser Ile Phe                                                           5                  10                                                                           SEQ ID NO:11,                           ______________________________________                                    

the fit is ambiguous because Thr was not detected after thecarboxypeptidase Y digestion. In contrast, a missing base at codon 47would introduce a frameshift resulting in a termination codon atposition 71. The predicted sequence of the C terminus of thispolypeptide would be

    ______________________________________                                        Ser Tyr Phe Gly Leu Arg Ile    SEQ ID NO:12,                                                      5                                                         ______________________________________                                    

which is a precise fit to the C-terminal sequence data for the fragment.

Deletion of T at codon 47 would also fortuitously introduce a novelrestriction site into the DNA by changing the base sequence from CCCGTGGto CCCGGG, the latter being the recognition sequence for the enzymeSmaI. Analytical digests were therefore performed on pIT4 and pCRP5 bydigesting 50-100 ng of each plasmid for 2-4 hours at 37° C. with EcoRI,SmaI, KpnI or SmaI and KpnI. The digestion products were separated on 1%agarose gels and visualized with ethidium bromide. Both plasmids weredigested with SmaI yielding fragments of the sizes expected if the T incodon 47 were missing. These results further confirm the results of theDNA sequence analysis. These results also demonstrate that the deletionhad occurred in the original plasmid pCRP5 and was not a result of thesubcloning process described in section A above. No undigested DNA wasdetectable in any of the plasmid digests.

Finally, a sample of the purified 7000 Mr fragment (purified asdescribed above) was submitted to Analytical Biotechnology Services forcomplete amino acid composition analysis. Briefly, the protein samplewas hydrolyzed in 6N HCl and derivatized with phenylisothiocyanate. Thephenylthiohydantoin-amino acids were detected by high performance liquidchromatography (HPLC). Once again, the results confirmed that there wasa base deletion in codon 47 which introduced a frameshift leading topremature termination of protein synthesis at codon 71.

The analysis of both the plasmid DNA and the Mr 7000 fragment, thus,provided convincing evidence that a base deletion in codon 47 wascausing premature termination of translation, and that the deletion hadoriginated in plasmid PCRP5. However, full-length mutant rCRP subunitswere being made (see FIG. 3).

A possible explanation for the synthesis of the full-length subunits wassuggested by a report in the literature [Tenchini et al., Inflammation,16, 93 (1992)]. Tenchini et al. describe a human CRP cDNA clone whichhad been isolated and sequenced. It had the base deletion in question incodon 47, but also contained a single base insertion at codon 64 (seeTable 2 below). Since the insertion occurs before the termination codonintroduced by the deletion, the proper reading frame is restored beforeprotein synthesis is terminated. The net result is a sixteen-amino acidsegment of out-of-frame sequence in the middle of the protein.

This segment differs greatly in amino acid composition from the knownCRP sequence, so amino acid analysis was performed on the purifiedmutant rCRP produced by pIT4. The protein was purified as follows.Mutant rCRP was purified using a two-step column procedure. Inclusionbody protein was solubilized, fractionated with ammonium sulfate anddissolved in urea as described above for the Mr 7000 fragment. Thismaterial was then applied to an S-Sepharose Fast Flow cation exchangecolumn. The column was developed with a 0-0.25M NaCl gradient in 6Murea, 25 mM sodium acetate, pH 4.0, to removed impurities. After theNaCl concentration was decreased to 0, the mutant rCRP was eluted with8M GuHCl, 25 mM sodium acetate, pH 4.0. The GuHCl eluate was thenapplied to a Superdex 200 gel filtration column (Pharmacia) which hadbeen equilibrated with 3M GuHCl, 4M urea in 25 mM sodium acetate, pH4.0. The purified protein was submitted to Analytical BiotechnologyServices for complete amino acid composition analysis. The results wereinconclusive.

Several additional experiments designed to detect the presence of aninserted base in codon 64 were negative. Moreover, the initial DNAsequence analysis of plasmid pIT4 had not detected the insertion of abase in codon 64 (see above). The combined experimental results,therefore, supported the conclusion that there is a base deletion incodon 47 and no base insertion prior to stop codon 71.

It is highly unlikely that the full-length rCRP subunits result fromoccasional read-through of the stop codon 71, since the frameshift dueto the deletion in codon 47 introduces a total of nine terminationcodons into the mRNA. Even if it were possible for the ribosomes to readthrough all nine stop codons, the frequency of the event should decreaseas the number of missed codons increases. This would generate apredictable ladder of products, with the largest being present in thelowest abundance. In contrast, only two predominant species have beenobserved, the 7000 Mr fragment encoded by the plasmid and thefull-length subunit (see FIG. 3).

Many explanations as to the source of the full-length rCRP have beeneliminated, and it is now believed that it results from a ribosomalframeshift whereby the ribosome shifts one or two bases duringtranslation of the mRNA so that the proper reading frame is restored.There are many documented examples of such events occurring in E. coli ,both as natural mechanisms for protein synthesis and as mutationsoccurring, for example, when an aminoacyl-tRNA is limiting. See, e.g.,Weiss et al., EMBO J., 7, 1503-1507 (1988); Tsuchihashi et al., Proc.Natl. Acad. Sci., 87, 2516-20 (1990); Gallant et al., J. Mol. Biol.,223, 31-40 (1992); Sipley et al., Proc. Natl. Acad. Sci., 90, 2315-19(1993); and Lindsley et al., Proc. Natl. Acad. Sci., 90, 5469-73 (1993).In the present case, the ribosomal frameshift must occur between the Tdeletion in codon 47 and the stop codon 71 to restore the reading frameand obtain a full-length product. The amino acid sequence of thefull-length rCRP coded for by codons 47-71 has been deduced (see sectionJ below).

    Table 2       - Amino acid sequence of CRP between residues 46-72 and corresponding     DNA sequence:      SEQ ID NO:13                                    Thr Arg Gly Tyr Ser Ile       Phe Ser Tyr Ala Thr Lys Arg Gln Asp Asn Glu Ile Leu Ile Phe Trp Ser     Lys Asp Ile Gly                        5                10                15          20      25                                                          ACC CGT GGG       TAC AGT ATT TTC TCG TAT GCC ACC AAG AGA CAA GAC AAT GAG ATT CTC ATA     TTT TGG TCT AAG GAT ATA GGA      81      SEQ ID NO:14                                                  Possible amino acid     sequence of Mr 7000 fragment between residues 46-72 assuming deletion     only and corresponding DNA sequence:      SEQ ID NO:15                                        Thr Arg Gly Thr Val       Phe Ser Arg Met Pro Pro Arg Asp Lys Thr Met Arg Phe Ser Tyr Phe Gly     Leu Arg Ile STOP                        5                10                15          20      25                                                          ACC CGG GGT       ACA GTA TTT TCT CGT ATG CCA CCA AGA GAC AAG ACA ATG AGA TTC TCA TAT     TTT GGT CTA AGG ATA TAG GA      80      SEQ ID NO:16                                                Tenchini et al. amino acid     sequence between residues 46-72 and corresponding DNA sequence:      SEQ ID NO:17                                     Thr Arg Gly Thr Val     Phe Ser Arg Met Pro Pro Arg Asp Lys Thr Met Arg Phe Phe Ile Phe Trp Ser     Lys Asp Ile Gly                        5                10                15          20      25                                                          ACC CGG GGT       ACA GTA TTT TCT CGT ATG CCA CCA AGA GAC AAG ACA ATG AGA TTC TTC ATA     TTT TGG TCT AAG GAT ATA GGA      81      SEQ ID NO:18     The nucleotides deleted or inserted are underlined when present. Amino     acids in boldface type are those differing from the sequence of CRP.

Clearly, the base deletion in codon 47 of the CRP coding sequence ofpIT4 was preventing efficient expression of rCRP, and mutagenesis wasnecessary to correct the sequence. In addition, some other features ofthe CRP coding sequence were identified which could hinder rCRPexpression. One of these is a potential stem-loop structure surroundingthe site of the deletion. A second is poor codon usage. The mutagenesis,therefore, had three goals: 1) to re-introduce the missing base intocodon 47; 2) to reduce the possibility of mRNA secondary structure; and3) to replace codons infrequently used in E. coli.

The mutagenic primer was designed with those goals in mind. The targetsequence for mutagenesis and the changes to be introduced are shownbelow. The bold letter indicates the base that was inserted into thesite of the deletion (codon 47). The underlined codons (48 and 50) areothers that were changed for purposes of increasing expression.

    ______________________________________                                        TARGET SEQUENCE:                                                                    CC TCG ACC CGG GGT ACA GTA TTT TCT CG     28                                            (SEQ ID NO:19)                                                  - DESIRED CHANGES:                                                                CC TCG ACC CGT GGT TAC AGC ATT TTC TCG    29                                               (SEQ ID NO:20)                                                     Gly 48:      Ser 50:                                                                        change codon change codon                                 usage, weaken usage                                                           2° structure                                                         ______________________________________                                    

Complementary oligonucleotide primers spanning the target sequence weresynthesized by the phosphoramidite method on an Eppendorf Synostat-Dsynthesizer using Eppendorf reagents. The sequences of these primers areshown as A and B in Table 3 below. Two additional oligonucleotides (Cand D) were synthesized for use as flanking primers. Their sequences andlocations relative to the CRP sequence and a schematic diagram of eachof the reactions performed are shown in FIG. 12.

                                      TABLE 3                                     __________________________________________________________________________    Pri-                                                                            mer Name Length Sequence Note                                               __________________________________________________________________________    C  Upstrm 1                                                                           25   CCCGCGAAATTAATACGACTCACTA                                                                       5' flanking                                         SEQ ID NO:21 primer                                                        B SmaI 29 CCTCGACCCGTGGTTACAGCATTTTC   Muta-genic                              upper  TCG primer,                                                              SEQ ID NO:20 puts T in                                                         and                                                                           removes                                                                       putative                                                                      stem loop                                                                 A SmaI 29 CGAGAAAATGCTGTAACCACGGGTC As above,                                  lower  GAGG but opposite                                                        SEQ ID NO:22 strand                                                        D BamHI 26 CTTTGTTAGCAGCCGGATCCGAGGT  3' flanking                              lower  A primer,                                                                SEQ ID NO:23 intro-duces                                                       Bam HI site                                                             __________________________________________________________________________

Reactions 1 and 2 illustrated in FIG. 12 employed 10 nmol ofHindIII-linearized pIT4 as template, 10 pmol of each primer, 0.2 mMdeoxynucleotide triphosphates (Pharmacia), 10 μl of 10x GeneAmp^(R)reaction buffer (Perkin Elmer Cetus), and 2.5 units AmpliTaq^(R) Taq DNAPolymerase (Perkin Elmer Cetus) in a total volume of 100 μl. Reactionbuffer contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂ and0.001% (w/v) gelatin (final concentrations). Reactions were carried outin a DNA Thermal Cycler Model 480 from Perkin Elmer Cetus. Following athree-minute incubation at 94° C., the unit was programmed to run 20cycles of 94° C., 1 min.; 50° C., 1 min.; 74° C., 1 min., ending with a7 min. incubation at 72° C.

PCR products were purified on a vertical 1.6% agarose gel (1.5 mm thick)run in TAE (Tris Acetate/EDTA). Prior to casting the gel, plates, combsand spacers were soaked in 1N HCl for 30 minutes to guard againstcontamination. The appropriate bands were excised and the PCR productspurified using a GeneClean II^(R) kit (Bio101 Inc.) according to themanufacturer's instructions. The DNAs were eluted in a volume of 100 μlTE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA). The yields of DNA were estimatedfrom the prep gel.

A second set of PCR reactions was performed to splice together thefirst-round products, thus restoring the full-length coding sequence.Fifty ng of the 3' product and 20 ng of the 5' product were used astemplate, and 10 pmol each of primers A and B were used foramplification. Other conditions for the reactions were as in the firstround. Four thermal cycles were performed prior to the addition of theprimers to give a chance for the extension reaction to begin. Afterprimers were added, the reactions were continued for another 20 cycles.

The vector pETV and the final PCR product were digested with NdeI andBamHI. The fragments were gel-purified using GeneClean. The vector wasthen treated with calf intestinal alkaline phosphatase. Vector andinsert were ligated, and the ligation mixture was used to transform E.coli BL21(DE3). Transformants were screened by minipreps performed asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(2nd ed., 1989, Cold Spring Harbor Laboratory Press). Analytical digestswere performed on the miniprep DNA using SmaI, NdeI and KpnI, NdeI andBamHI, and XbaI and HindIII. The products were separated on a 1% agarosegel and visualized with ethidium bromide. The DNA was demonstrated to berecombinant without the SmaI site. Subsequent sequence analysisconfirmed that the CRP coding sequence had the expected sequence withall of the intended changes incorporated (see section G below); noerrors in the sequence were detected. This plasmid was designated pIT10.

G. Preparation Of pIT13

Novagen, Inc. has developed several variants of the BL21(DE3) expressionstrain and of the pET3a vector used in the work described above. E. colistrain BLR(DE3) is identical to BL21(DE3), except that it is rec A" andis therefore recombination deficient. Plasmid pET-24a(+) is a derivativeof plasmid pET-3a in which the β-lactamase gene has been replaced withthe gene conferring resistance to kanamycin. pET-24a(+) possesses the T7promoter and has the lac operator sequence inserted between thispromoter and the multiple cloning site. This vector is designed toprovide a binding site for the lac repressor which will, in the absenceof inducer, prevent transcription of the target gene.

Since pET-24a(+) is a derivative of pET-3a, it has the XbaI and BamIsites in the same positions as in the pETV vector used in the previousexpression system. The mutant CRP coding sequence was therefore excisedfrom pIT10 and cloned into pET-24a(+) digested with the same enzymes.The ligation mixture was transformed into E. coli DH5α cells, andtransformants were selected by kanamycin resistance. Colonies werescreened by restriction analysis of miniprep DNA (procedure describedabove in section F). Plasmid DNAs from confirmed recombinants werepurified on cesium chloride gradients. One such recombinant wasdesignated pIT13. The preparation of pIT13 is illustrated in FIG. 13.

Expression plasmid pIT13 was transformed into E. coli BLR(DE3).Transformants were selected by resistance to kanamycin. The combinationof the kanamycin resistance marker and the T7-lac operator promoter helpto ensure that the plasmid will remain under selection and that verylittle, if any, expression will occur prior to induction. The rec A⁻genotype of the strain limits the possibility of recombination. Finally,the characteristics of the strain permit ease of handling on aproduction scale.

Culturing the pIT13/BLR(DE3) strain under the same conditions as thepIT10/BL21(DE3) strain (except for the type of antibiotic) resulted inthe production of 12.5 g of inclusion body protein for thepIT13/BLR(DE3) strain as compared to 8.0 g for the pIT10/BL21(DE3)strain. Expression and purification of mutant rCRP from pIT13 in E. coliBLR(DE3) are further described in the next section.

Plasmid pIT13 was isolated from the E. coli BLR(DE3) expression strainand purified on a cesium chloride gradient. The CRP coding region wassequenced in the expression vector by the dideoxy chain terminatormethod of Sanger et al., Proc. Nat'l. Acad. Sci. USA, 74, 5463-5467(1977) using Sequence version 2.0 (U.S. Biochemical Corp.) and ³³ P-DATPas the radiolabel.

Reaction products were separated on a 5% Hydrolink Long Ranger gel (ATBiochem Corp.). A sample of each reaction mixture was loaded andelectrophoresed until the bromphenol blue dye marker reached the bottomof the gel (1st load). Identical samples were then applied to theremaining wells (2nd load) and electrophoresis continued until thebromphenol blue dye marker contained in the second set of samplesreached the bottom of the gel. The gel was then dried and contacted withX-ray film for 2, 6.5, or 17 hours, and the sequence was read from thedeveloped film.

The sequence determined by this procedure corresponded exactly to thecoding sequence reported by Woo et al., J. Biol. Chem., 260, 13384-88(1985) with the following differences: the synonymous substitution inthe wobble position of codon 152 (see section F above); the deletion ofthe leader sequence and introduction of an ATG codon for translationinitiation; the substitution of a T for the G in the wobble position ofcodon 48; the substitution of a C for the T in the wobble position ofcodon 50; the substitution of a CGC codon (alanine) for the TGC codonfor cysteine 36; and the substitution of a CGC codon (alanine) for theTGT codon for cysteine 97. It was, therefore, concluded that thepredicted amino acid sequence of the mutant rCRP is identical to thesequence reported for unmutated CRP, except for the three changespurposefully factored into the molecule: 1) the N-terminal PCA aminoacid of unmutated CRP is preceded by a formylated-methionine and isitself changed to glutamine; 2) cysteine residue number 36 issubstituted with an alanine residue; and 3) cysteine residue number 97is substituted with an alanine residue.

The DNA sequence results demonstrate that the genetic manipulationsperformed on the CRP coding sequence of pIT4 to correct the sequence andto obtain better expression of the mutant rCRP were performed correctly.No base additions, deletions, or substitutions were unintentionallyintroduced into the sequence.

H. Expression And Purification Of Mutant rCRP

The bacterial fermentations and the preparation of inclusion bodies wereperformed by Bio-Technical Resources (Manitowoc, Wis.) under asepticconditions following the Good Laboratory Practices (GLP) guidelines ofthe Food and Drug Administration. Briefly, E. coli BLR(DE3) bearing thepIT13 plasmid was cultured in a 250 liter pilot fermentor (New BrunswickScientific) as follows. E. coli BLR(DE3) cells were grown at 37° C. in afourteen-liter New Brunswick Model FS614 fermentor in a mediumcontaining NZ amine A^(R) (enzymatic hydrolysate of casein; QuestInternational; 20 g/l), (NH₄)₂ SO₄ (2 g/l), KH₂ PO₄ (1.6 g/l), Na₂ HPO₄:7H₂ O (9.9 g/l), sodium citrate (0.65 g/l), MgSO₄ (0.24 g/l), glucose(22 g/l) and 50 μg/ml kanamycin. The culture was aerated usingcompressed air at 50 liters per minute. Synthesis of the mutant rCRP wasinduced with 1 mM IPTG (Gold Biotechnology Inc.) when the cell densityreached OD₆₀₀ =5. After a three-hour induction period, the cells wereharvested by continuous flow centrifugation at 15,000 rpm in a Sharplesmodel AS16VB tubular bowl centrifuge. The harvested cell paste wastransferred into sterile plastic bags and frozen at -80° C.

The cell paste was thawed by placing the sealed plastic bags in a 45° C.water bath. The thawed cell paste was then suspended in cold breakagebuffer (20 mM Tris-HCl, pH 7.6, 5 mM EDTA, 1 mM phenylmethyl sulfonylfluoride (PMSF; Sigma Chemical Co.) at a ratio of 200 ml buffer per gramcell paste and processed in a sterile blender at low speed for 30-45seconds. The homogeneous suspension was passed twice through a NyroSoave homogenizer (equipped with a cell disruption valve) at a pressureof 500 bar. As it exited the homogenizer, the suspension was passedthrough a sterile cooling coil packed in an ice bath for cooling. It wasthen collected in a sterile covered container on ice.

This lysate was centrifuged at 12,000 x g in a Beckman model J2-21centrifuge for 10 minutes at 4° C. The pellet was resuspended inbreakage buffer at 100 ml buffer per gram of initial cell paste. Thesuspension was then passed twice through the homogenizer, this time at700 bar. The extract was centrifuged at 12,000 x g for 25 minutes at 4°C. in a Beckman model J2-21 centrifuge to collect the inclusion bodypellet.

The inclusion bodies were washed once with breakage buffer at a ratio of150-200 ml buffer per 100 grams of initial cell paste by suspending thepellet in the buffer with a sterile glass rod and then centrifuging asdescribed above. They were then washed three times with wash buffer(breakage buffer containing 0.5% Triton X-100 (Sigma Chemical Co.) at aratio of 100-150 ml per 100 grams initial cell paste. The pellets werefinally suspended in approximately 75 ml of breakage buffer per 100grams of initial cell paste, aliquoted into sterile tubes, and frozen at-80° C. until further processing.

The concentration of protein in the inclusion body suspension wasestimated using the Bicinchoninic acid protein assay (Pierce ChemicalCo.) or by solubilizing the protein in 6M GuHCl and measuring the A₂₈₀(using the extinction coefficient for pure mCRP; 1.95[mg/ml]⁻¹). Theinclusion bodies were then pelleted by centrifuging for 10 minutes at12,000 rpm in a Sorvall GSA rotor. The supernatant was discarded, andthe pellet was dissolved in a solution of 6M GuHCl in 25 mM Tris, pH 8(6M GuHCl/Tris), to a concentration of between 5-10 mg protein/ml. Theconcentration of solubilized protein was determined by measuring theA₂₈₀ of a diluted sample. The inclusion body preparation was thendiluted to a final concentration of 5 mg protein per ml with the 6MGuHCl/Tris buffer.

Once the protein had been solubilized and diluted as just described, aninitial ammonium sulfate fractionation step was performed. Using aperistaltic pump, a saturated solution of ammonium sulfate was addeddropwise with stirring to a final concentration of 25% saturation at 0°C. The resulting suspension was stirred on ice for another 30 minfollowing the completion of the ammonium sulfate addition, after whichit was centrifuged 10 min at 12,000 rpm in a Sorvall GSA rotor. Thesupernatant, which contains primarily impurities, was discarded. Thepellets were washed with sterile saline in a volume equivalent to thatof the original solubilized protein solution to remove residual ammoniumsulfate. Pellets were stored at 4° C. until further processing.

A Q-Sepharose Fast Flow^(R) anion exchange column (Pharmacia) wasequilibrated with 25 mM Tris-Cl, pH 8. The washed ammonium sulfateprecipitate containing the mutant rCRP was solubilized at aconcentration of 0.5 mg/ml by adding 10 mM Tris base and stirring untila suspension was formed. NaOH was then added until the solution becameclear (pH 12.2-12.5). The pH was then titrated to 9.0 with HCl. Thesolubilized protein was loaded onto the Q-Sepharose Fast Flow^(R) columnat a linear flow rate of 30 cm/hour, with the total protein load notexceeding 5 mg/ml of resin. The column was washed with 1 volume of theequilibration buffer (25 mM Tris-Cl, pH 8), and then developed with atwo-column volume linear gradient of 0-1M NaCl in 25 mM Tris-Cl, pH 8.After the NaCl concentration was rapidly returned to 0, the column waswashed with an additional column volume of the equilibration buffer. Themutant rCRP was finally eluted from the column with 8M GuHCl in 25 mMTris, pH 8. A₂₈₀ was measured using the BioPilot^(R) system.

The mutant rCRP adsorbs very strongly to the Q-Sepharose Fast Flow^(R)column in the absence of a denaturant and will not elute with the NaClgradient or with a pH gradient up to at least pH 12, allowing for anexcellent separation of the mutant rCRP from endotoxin. The mutant rCRPreadily elutes from the column in the presence of a denaturant, such asGuHCl. However, urea should not be used for this elution step.

The eluate from the Q-Sepharose Fast Flow^(R) column was concentrated byprecipitation with 25% saturated ammonium sulfate as described above.After the pellet was washed with saline, it was dissolved in 3% (w/v)sodium dodecyl sulfate (SDS), 25 mM Tris-Cl, pH 9.0 at a concentrationof 5-10 mg protein/ml (estimated using absorbance at 280 nm andextinction coefficient for pure mCRP). It was then applied to a 5 cm×92cm Superdex 200 gel filtration column previously equilibrated in 1% SDS,25 mM Tris-Cl, pH 8.0. The sample size was 1-1.5% of the column volume,and the linear flow rate was 30 cm/hr. The mutant rCRP typically elutedat a volume of 1120 ml (60% of the total bed volume), and collectionbegan when the peak reached 25-30% of its expected height and wasterminated when it returned to the same level. A₂₈₀ was measured on theBiopilot^(R) system.

The mutant rCRP collected from the Superdex 200 column were firstchilled to 0-4° C. Any precipitated SDS was then removed by a 5-10 mincentrifugation at 12,000 rpm in a Sorvall GSA rotor. Then 5 μl of a 25%(w/v) solution of KCl per ml of supernatant was added, and the insolublepotassium dodecyl sulfate was removed by centrifugation at 12,000 rpm ina Sorvall GSA rotor. The KCl addition and centrifugation were repeatedtwice more. Finally, the supernatant was brought to 30% saturatedammonium sulfate and the resulting precipitate was collected bycentrifugation at 12,000 rpm in a Sorvall GSA rotor. The pellet wasdissolved at 3-4 mg protein/ml (estimated as described above) in 10 mMTris-Cl, pH 9.2.

The solubilized mutant rCRP was next applied to a Sephadex G-25 (fine)column (Pharmacia) equilibrated in 10 mM Tris-Cl, pH 7.4, at a linearflow rate of 70 cm/hr with the sample volume not to exceed 20% of thecolumn volume. A₂₈₀ and conductivity were measured on the Biopilot^(R)system, and the mutant rCRP was usually present at a concentration of1.5-2 mg/ml.

FIG. 14 presents the SDS-PAGE results for the complete purificationscheme and illustrates the increase in purity attained in each step. Thefinal mutant rCRP preparation is nearly homogenous. To perform theSDS-PAGE, samples were boiled in 2.5% SDS and 5% (v/v)β-mercaptoethanol, and 8 μg of each sample was loaded onto a 20%homogeneous Phast gel. Proteins were visualized by staining withCoomassie Brilliant Blue.

The concentration of residual SDS in the mutant rCRP preparation wasmeasured using the acridine orange binding assay described in Anal.Biochem., 118, 138-141 (1981). Following the purification protocoldescribed above, the concentration of residual SDS in the mutant rCRPpreparation is routinely below the limit of detection (˜0.001 w/v) ofthe assay.

Finally, the purified mutant rCRP was sterile filtered through 0.2 μfilters. The filtered, sterile mutant rCRP was then bottled inpyrogen-free sterile vials for injection.

I. Characterization Of The Mutant rCRP

1. SDS-PAGE and Western blot

Purified mutant rCRP produced by the method described in section H wascompared with mCRP in SDS-PAGE and Western blot analyses. SDS-PAGE wasperformed using 20% Phast gels (Pharmacia). Gels were stained forprotein using Coomassie Brilliant Blue. Gels were subjected to Westernblot analysis by transferring the electrophoretically-separated proteinonto nitrocellulose paper and adding monospecific goat anti-neo-CRPantiserum (prepared as described in section D above).

The results are shown in FIGS. 15A and 15B. In particular, the mutantrCRP and mCRP migrated as single bands on SDS-PAGE; no extraneousCoomassie-staining bands were noted in either sample suggesting thatboth the mCRP and the mutant rCRP are pure to the level of sensitivityof this analytical procedure. Each protein migrated with identicalapparent molecular weights of Mr 23,000. This agrees with the literaturevalues for the molecular weight of the unmutated CRP subunit in SDS-PAGEand with the molecular weights calculated from the known primarysequences of the unmutated CRP subunit (Mr 22,976) and mutant rCRP (Mr23,114). These data show that the mutant rCRP has a molecular weight andpurity essentially identical to mCRP.

Western blot analysis using mCRP-specific monoclonal antibody 3H12demonstrates that the protein bands of both the mutant rCRP and of mCRPstrongly reacted with this antibody (see FIG. 15B). No additional bandsappeared in the transblotted lanes of either isolated protein,indicating an even greater level of purity than indicated by theSDS-PAGE results, since the Western blot results are more sensitive thanthe Coomassie staining technique. Monoclonal antibody 3H12 is known toreact with the carboxy-terminal octapeptide of mCRP. These data showthat the mutant rCRP expresses a very similar (probably identical)epitope as that of mCRP.

2. ELISA Analyses

An ELISA assay was performed to determine if monoclonal antibodiesspecific for different epitopes on mCRP would react with the mutant rCRPproduced by pIT13 and purified as described in section H. The ELISAassay was performed as described in section D above using the followingthree monoclonal antibodies: 3H12, 8C10 and 7A8. The preparation andproperties of mAb 3H12 and 8C10 are described above in section D. Thepreparation and properties of mAb 7A8 are described in U.S. Pat. No.5,272,257, PCT application Wo 91/00872, Ying et al., J. Immunol., 143,221-228 (1989) and Ying et al., Mol. Immunol., 29, 677-87 (1992).Monoclonal antibody 3H12 reacts with the terminal octa-peptide of mCRP,as noted above. Monoclonal antibody 8C10 reacts with an epitope near theamino end of the mCRP sequence; this epitope is presumed to involveresidues 22-45, which includes cysteine 36 which is mutated in themutant rCRP. See Ying et al., Mol. Immunol., 29, 677-687 (1992).Monoclonal antibody 7A8 reacts with a third region of mCRP presumed toinvolve residues 130-138. See Ying et al., Mol. Immunol., 29, 677-687(1992).

The ELISA results are shown in FIGS. 16A-C. All three monoclonalantibodies reacted equivalently with mCRP and the mutant rCRP producedby pIT13, including mAb 8C10 which reacts with an epitope believed toinclude cysteine 36. Each binding curve had a similar shape and relativeintensity, suggesting that the mutant rCRP and mCRP both are comprisedof, and express, these three distinct epitopes.

J. Amino Acid Sequence Of Mutant rCRP Produced By pIT4

Experiments were conducted to determine the amino acid sequence of thefull-length mutant rCRP subunit produced by pIT4. Since this full-lengthproduct is believed to be produced by a ribosomal frameshift duringtranslation of the mRNA, its amino acid sequence cannot be determinedfrom the DNA sequence of pIT4 (see section F above).

First, mCRP and the full-length mutant rCRP subunits produced by pIT4and pIT13 were digested with2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-Skatole;Pierce Biochemicals) which cleaves on the C-terminal side of tryptophanresidues. The full-length products of pIT4 and pIT13 were produced byculturing E. coli BL21(DE3) and BLR(DE3), respectively, and purifyingthe full-length products from the cultures, all as described above. ThemCRP and the final purified pIT4 and pIT13 products were solubilized ata concentration of 50 mg/ml in 25 mM Tris-Cl, pH 8, containing 6M GuHC1.The reactions were initiated by the addition of four volumes of glacialacetic acid containing 25 mg/ml BNPS-Skatole. Thus, the final reactionconditions were 10 mg/ml protein, 1.2M GuHCl, 80% acetic acid and 20mg/ml BNPS-Skatole. After a 24-hour digest at room temperature in thedark, 42.5 μl of each sample were transferred to an Eppendorf tube, andan equal volume of saturated ammonium sulfate was added to each tube.After a two-minute centrifugation in an eppendorf microcentrifuge, thesupernatants were discarded, and the pellets were washed with 200 μl of0.1M Tris-Cl, pH 7.4. The pellets were finally suspended in 42.5 μl of8M urea in 25 mM Tris-Cl, pH 8.5. The samples were then analyzed bySDS-PAGE performed as described in section I above.

There is a Trp in the CRP sequence at position 67 which is also found inthe Tenchini et al. sequence (see Table 2). Cleavage at this site wouldproduce a fragment of ˜8000 Mr. Digestion of the mCRP and of the pIT13product produced a prominent fragment of this size, whereas digestion ofthe pIT4 product produced a fragment of ˜11,000 Mr, the expected size ifTrp68 were absent. Since the pIT4 and pIT13 products were digested underidentical conditions, these results strongly suggest that Trp68 ismissing in the pIT4 product and that the out-of-frame sequence of pIT4extends at least to codon 67.

In a second experiment, the two mutant rCRP's were digested withendoproteinase LysC (Boehringer Mannheim Biochemicals), which cleavesspecifically at the C-terminal side of lysine residues. The purifiedmutant rCRP's (prepared as described above, this section) weresolubilized in 8M urea, 25 mM Tris-Cl, pH 8.0, at a concentration of 5mg/ml. Then, 0.1M Tris-Cl, pH 7.4, enzyme and water were added insufficient quantities to give final conditions of 25 mM Tris-Cl, pH 7.4,4M urea, 0.05 U enzyme, and 2.5 mg/ml protein. The digests wereincubated for 24 hours at room temperature, and the digestion productswere analyzed by SDS-PAGE as described in section I above, except thathigh density gels designed for peptide separations were used.

In the region of the protein sequence in question, digestion of thepIT13 product with LysC would be expected to yield fragments of Mr 2961(residues 32-57), 1548 (residues 58-69) and 4827 (residues 70-114). Inthe pIT4 product, the lysine at position 57 is predicted to be absent,and a novel lysine present at position 59 (see Table 2). If the lysineat position 69 is present in this protein, digestion with LysC shouldproduce fragments of Mr˜3200 (residues 32-59), ˜1100 (residues 60-69)and 4827 (residues 70-114). In contrast, if it is absent, the 4827 and˜1100 Mr fragments would be replaced by a fragment of ˜6000 Mr. Afragment produced by digesting both the pIT4 and pIT13 proteins whichmigrates at ˜4800 Mr was observed, and there is no trace of a ˜6000 Mrfragment in the pIT4 protein digest. These results indicate that thereis a lysine at position 69 in the mutant rCRP's produced by both pIT4and pIT13.

Taken together with the original amino acid composition and DNA sequencedata (see section F above), the results of these two experimentsstrongly suggest that the sequence of the full-length mutant rCRPproduced by pIT4 has the following amino acid sequence from position 47to position 69:

    ______________________________________                                        Arg Gly Thr Val Phe Ser Arg Met                                                                  5                                                             - Pro Pro Arg Asp Lys Thr Met Arg                                                 10                  15                                                    - Phe Ser Tyr Phe Gly Leu Lys                                                             20                                                                -                   SEQ ID NO:24                                           ______________________________________                                    

Residues shown in bold are those that likely differ from the bona fideCRP sequence and from the Tenchini et al. sequence (see Table 2 above).Those residues which are underlined are still questionable in terms oftheir identity.

Example 3 Effect of mCRP on Megakaryocytopoiesis in Vitro

Six to seven week old specific pathogen free ICR mice were obtained fromCLEA Japan, Ltd., Tokyo, Japan, and climatized for three weeks. Bonemarrow cells were then harvested from mice femurs.

Meg-CFC cultures were performed according to the procedure described inMizoguchi et al., Exp. Hematol., 7:346 (1979). The bone marrow cells(5×10⁵ /dish) were immobilized in a plasma clot attached to the bottomof a Petrie dish. Plasma clots were formed by mixing bone marrow cellsuspensions in Eagle's minimal essential medium/Hanks' balanced saltsolution (HMEM from GIBCO) containing 20% fetal calf serum, 20% NCTC109solution (DIFCO), 10% bovine embryo extract (diluted 1:5), 1% bovineserum albumin, 0.02 mg/ml asparagine, 7% Pokeweed-mitogen stimulatedspleen lymphocyte-conditioned medium and 10% bovine citrated plasma(Sigma) to make a final volume of 0.4 ml-0.6 ml per dish. Afterclotting, 0.4-0.6 ml HMEM plus 10% fetal calf serum was added to eachPetrie dish.

The bone marrow cultures were incubated in the presence of eithercontrol solutions or solutions containing various doses of either mCRPor native CRP. Both soluble and suspended forms of mCRP were preparedusing partially purified native CRP (Western States Plasma, Fallbrook,Calif.) as the starting material. The native CRP was sterile filteredand further purified by ion exchange chromatography as described inExample 1, Section A. The solution and suspended forms of mCRP wereproduced by processing the native CRP in accordance with the methodsdescribed in Example 1, Section B.

The control solutions generally were identical buffers except lackingmCRP or native CRP. In some experiments, control solutions consisted ofbuffers containing other hematopoietically-active cytokines such asG-CSF or IL-6. The test cultures were incubated with either native CRP,solution-mCRP or suspension-mCRP. Native CRP and mCRP were tested atconcentrations ranging from 0.1 μg to 100 μg/ml.

The bone marrow cultures were incubated for 5 days at 37° C. in a fullyhumidified incubator containing 5% CO₂. The plasma clots were thendehydrated, fixed with 5% glutaraldehyde, stained foracetylcholinesterase activity and counter-stained with Harrishematoxylin. Next, the cultures were analyzed using an invertedmicroscope. Colonies consisting of four or more positively-stained cellswere scored as one colony.

At doses of 2.5 μg to 20 μg, mCRP increased the number of megakaryocytecolonies formed, increasing from approximately 100 colonies/2×10⁵ bonemarrow cells to approximately 180 colonies/2×10⁵ bone marrow cells. (SeeFIG. 5A). The mCRP-treated cultures showed megakaryocyte colonyformation from 40-to-100% above that observed in control cultures andcultures incubated with native CRP. Native CRP-treated cultures showedthe same number of colonies/dish as the control, buffer-treated samples.

FIG. 5B illustrates the kinetics of that megakaryocyte colony formation.Control megakaryocyte cultures appeared on day 2 as immature cells, andon day 3 as more mature cells. Maximum numbers of colonies were noted ondays 5-6. After this time, the cytoplasm of the megakaryocyte coloniesdecreased indicating degradation of colonies. Modified-CRP-treatedmegakaryocyte colonies were more mature on day 4 compared to controlcolonies. The number of colonies quantified in mCRP-treated culturesincreased approximately 40% (p<0.01) on days 5-6 compared to controlcultures. In this assay system, IL-6 showed no effect onmegakaryocytopoiesis at doses from 10 μg to 100 μg/dish.

Under the microscope, the bone marrow cells treated with mCRP showedunusual proliferation with unusual shape. (See FIGS. 6A and 6B).Modified-CRP-treated megakaryocytes appeared larger than those observedin control cultures, with more pronounced pseudopods and heavily stainedcytoplasm. The intensity of cytoplasmic staining is usually interpretedas an indication of megakaryocyte maturity. The data thus suggests thatmCRP promoted megakaryocyte maturation and demonstratesMegakaryocyte-Potentiator activity.

Example 4 In Vivo Effect of mCRP on Thrombocytopoiesis in Normal Mice

Six to seven week old specific pathogen free ICR male mice were obtainedfrom CLEA Japan, Ltd., Tokyo, Japan and climatized for a minimum of oneweek. Four groups of mice consisting of 4 mice/group or 5 mice/groupwere utilized in the study. Each group received either control buffer,native CRP, mCRP (in solution), or MCRP (in suspension). The mCRP wasprepared using partially purified native CRP (Western States Plasma,Fallbrook, Calif.) as the starting material. The native CRP was sterilefiltered and further purified by ion exchange chromatography asdescribed in Example 1, Section A. The solution and suspended forms ofMCRP were produced by processing the native CRP in accordance with themethods described in Example 1, Section B.

A total of 100 μg of protein (approximately 2-4 mg/kg body weight) wasinjected daily for 5 consecutive days (Day 1 through Day 5). Theinjections were made subcutaneously (2 injections/day; FIGS. 7A and 7B)or intraperitoneally (1 injection/day; FIG. 7C). Control mice receivedeither control buffer lacking protein or sterile saline.

Blood analyses for the mice were performed by collecting 10 or 20 μl ofblood from each mouse by slightly cutting the tail vein with a sharprazor. For each analysis, the first 10 μl of blood was wiped away toavoid tissue fluid. The blood sample was collected in a DrummondMicrodispenser and diluted into a buffered solution containing achelating agent (commercially available from Sysmex F800 vendor).Leukocyte (WBC) numbers, erythrocyte (RBC) numbers, mean erythrocytevolume (MCV), platelet (PLT) numbers, mean platelet volume (MPV),concentration of hemoglobin, and hematocrit were measured at varioustime intervals. A semi-automatic blood cell counter F800 (Sysmex) wasused to make these measurements.

By day 6, mice injected subcutaneously with solution mCRP showedapproximately a 25-30% increase in blood platelet levels compared tocontrols (FIG. 7A; p<0.005) and a significant increase in platelet mass(FIG. 7B; p<0.001). As discussed in the Background section, it has beenreported that platelet count and mean platelet volume (MPV) areinversely related, i.e., as platelet numbers increase, MPV decreases[Thompson et al., Blood, 72:1-8 (1988)]. By taking the product of theplatelet number and the MPV, a parameter termed "platelet mass" isdefined. It is presently believed that platelet mass may correlate moreclosely with platelet function than does the platelet count alone.Therefore, calculation of platelet mass may be a superior measurementindicating the "maturity" and "function" of circulating platelets.

Blood platelet levels fell to control levels 1-2 weeks after therapy wasstopped. Similar results were achieved when suspended mCRP wasadministered subcutaneously (data not shown).

When solution mCRP was administered intraperitoneally, thethrombopoietic activity again increased to significant levels by day 6(p<0.05) compared to control groups (FIG. 7C). The increase in bloodplatelets in mCRP-treated mice persisted over the next two weeks (to day19). Administering suspension mCRP intraperitoneally showed a similarsignificant increase in blood platelet number (p<0.05 on day 19) andplatelet mass (p<0.05 on day 19). Native CRP-treated animals showedvariable results (data not shown).

Example 5 In Vivo Effect of mCRP on Thrombocytopoiesis inCyclophosphamide-treated Mice

Modified-CRP was prepared using partially purified native CRP (WesternStates Plasma, Fallbrook, Calif.) as the starting material. The nativeCRP was sterile filtered and further purified by ion exchangechromatography as described in Example 1, Section A. Solution mCRP wasthen produced by processing the native CRP in accordance with themethods described in Example 1, Section B. Mice were injectedsubcutaneously with the solution mCRP at a dose of approximately 3 mg/kgbody weight once/day on Days -4, -3, and -2. On Day zero, the mice wereinjected intraperitoneally with the chemotherapeutic drug,cyclophosphamide, at a dose of 250 mg/kg of body weight. Blood plateletlevels were depressed by this amount of drug so that on Day 4, plateletcounts fell to about half of the pre-treated levels. After chemotherapywas administered, the solution mCRP was injected subcutaneously in themice at a dose of approximately 3 mg/kg body weight once/day for 10days. Control animals were injected similarly with bovine serum albumin("BSA") solution.

The results are illustrated in FIGS. 8A-8D. Treatment with solution mCRPresulted in accelerated recovery of platelet counts so that on day 7,the counts were near 90% of the starting level (See FIG. 8A). Controlanimals, receiving BSA solution instead of solution mCRP, took at leastanother two days to reach that level. On day 7, the control animals hadonly recovered to approximately 65% of pre-treatment platelet levels.Using the two-tailed students t-test, the platelet level of test animalson day 7 was found to be significantly increased over control mice to ap value <0.005.

Example 6 In Vivo Effect of mCRP on Thrombocytopoiesis in X-irradiatedMice

ICR normal mice (three treatment groups, consisting of 4-5 mice/group)were X-irradiated with a dose of 2.0 Gy (200 kV, 20 mM, 0.5 mm Cu/0.5 mmAl filters, dose rate 72 cGy/min) (Gy=Gray, 1 gray is equivalent to 100Rads). At twenty hours and at two hours before completion of theirradiation, and once/day for 10 days after irradiation treatment wasstopped, the animals were injected subcutaneously with approximately 3mg/kg body weight solution mCRP. The solution mCRP was prepared usingpartially purified native CRP (Western States Plasma, Fallbrook, Calif.)as the starting material. The native CRP was sterile filtered andfurther purified by ion exchange chromatography as described in Example1, Section A. The solution mCRP was then produced by processing thenative CRP in accordance with the methods described in Example 1,Section B. Control animals were similarly injected with bovine serumalbumin (BSA) solution.

The results are illustrated in FIGS. 9A-9D. Treatment with solution mCRPresulted in accelerated recovery of platelet counts so that by day 13after X-irradiation, the counts were near 100% of the starting level(See FIG. 9A). Control animals, receiving BSA solution instead ofsolution mCRP, took at least another two days to reach that level. Onday 13, control animals had only recovered to about 70% ofpre-irradiation levels. Using the two tailed student's t-test, theplatelet level of test animals was found to be significantly increasedover the control animals on day 13 to a p value of <0.005 and on day 15to a p value of <0.025.

Example 7 In Vivo Effect of mCRP on Thrombocytopoiesis in SIV-infectedRhesus Monkeys

Two male rhesus monkeys infected with the Simeon Immunodeficiency Virus(SIV) were treated with approximately 2.8 mg/kg body weight/day ofsuspension mCRP by bolus intravenous injection on days 1, 2, 3, 4, and 5of a three week period. The suspension mCRP was prepared as described inExample 1. Blood samples were drawn just prior to administration of mCRPon days 1, 3, and 5. Additional data points were also collected on days12 and 19.

On days 12 and 19, both monkeys showed between 33 and 96% increase inblood platelet counts over pre-infusion values.

Example 8 In Vivo Effect of mCRP on Thrombocytopoiesis in HIV-positiveHumans

Three HIV-positive male patients each received 7 infusions of suspensionmCRP at a dose of 3 mg/kg body weight over a period of 15-29 days. Thesuspension mCRP was prepared using partially purified native CRP(Western States Plasma, Fallbrook, Calif.) as the starting material. Thenative CRP was sterile filtered and further purified by ion exchangechromatography as described in Example 1, Section A. The suspended mCRPwas then prepared by heating the native CRP to 60° C. for 1 hour in thepresence of 1 mM EDTA. The patients received intensive mCRP therapyduring the first week (from 3-to-5 injections each), followed byonce-a-week infusions for the next two-to-four weeks. Data was gatheredover a period of two-to-three months before, during, and after theinfusion of mCRP.

Overall, multiple infusions of mCRP were tolerated well with nosignificant adverse effects. A fever response was noted after allinfusions; this was controlled by giving each patient aspirin andTylenol®. No sign of organ damage (kidney, liver, lung pancreas, muscle)was noted, and no adverse effect on reticulocytes or leukocytes wasnoted. There was a drop in the hematocrit of each patient. However, thiswas not deemed related to MCRP therapy. Total lymphocyte counts,CD4+lymphocyte counts and CD8+ lymphocyte counts, increased in allpatients correlating with mCRP therapy. Serum electrolytes, enzymes,proteins and other factors (e.g., glucose, triglycerides, cholesterol,uric acid, BUN, creatinine) all remained within normal limits throughoutthe study. Both intrinsic and extrinsic coagulation pathways wereunaffected by intravenous infusion of mCRP.

All three patients showed an increase in blood platelet levels after theperiod of intensive mCRP therapy. This increase persisted throughout theexperimental period. All three patients showed an initial dip in bloodplatelet levels (approximately 20-30% drop) during the period ofintensive mCRP therapy (days 2-6). (See FIGS. 10A and 10B). This dropwas short lived, however, as a rapid increase was observed in allpatients on day 8. All three patients showed from 25-to-50% increases inblood platelet counts compared to pre-infusion averages. (See FIGS.10C-10E).

FIGS. 11A and 11B illustrate that the mCRP therapy increased plateletmass in all three HIV positive patients involved in the study. In thefirst 3-to-5 days of mCRP therapy, there was a slight decrease in bloodplatelet counts but no change, or increase in MPV (data not shown).Typically, it takes approximately 3 days for the process ofmegakaryocytopoiesis to occur. After 3-5 days of mCRP therapy, plateletmass increased 20-40% in these three patients.

The data suggests that early on (in the first 3-5 days of mCRP therapy),there is a slight decrease in blood platelet counts, and no change orincrease in the mean platelet volume. After 3-5 days, the time it takesto stimulate megakaryocytes to produce more platelets, there is anincrease in platelet number in the blood. These new platelets are ofnormal size, and are not smaller as would be expected. This suggeststhat mCRP may function like a Megakaryocyte-Potentiator factor,contributing not only to increase in platelet numbers, but to plateletmass as well.

Example 9 The Effect Of mCRP and Mutant rCRP On Human Bone MarrowMegakaryocytopoiesis

Low-density non-adherent mononuclear cells were prepared from bonemarrow from patients undergoing total hip arthroplasty. Briefly, asample containing bits of bone and surgical debris was added toMEM-alpha Eagle Medium (Gibco) supplemented with heparin, EDTA and 5μg/ml sterile DNase I (Boehringer Mannheim). The sample was gentlyshaken and incubated for 5 minutes at 0° C. The supernatant containingthe bone marrow cells was collected, and the residual dense bonematerial was reextracted multiple times until the bone appeared white.All supernatants were pooled. Cell aggregates were broken up by passingthe recovered cell suspension gently through an 18 guage needle.

The cell suspension was layered on top of an equal volume of 1.077 g/LFicoll-Paque (Pharmacia) and the mixture was centrifuged using aswinging bucket rotor at 400 x g for 30 minutes at ambient temperature.The white layer of cells which collected at the interface between theFicoll-Paque and the medium is referred to as low-density mononuclearcells.

Cells were collected, suspended in IMBM medium (Difco) and were washedmultiple times before being resuspended in a minimal volume of IMBMmedium. Cells were then counted and were diluted to 1×10⁶ cells/ml inIMBM medium supplemented with 10% Gold-Fetal Bovine Serum (ICN). Thecells were then added to a plastic dish and were incubated for from 2hours to overnight at 37° C. in a 5% CO₂ environment to remove adherentcells. The supernatant, which contains low density mononuclear cells(MNC's) was collected and washed in IMBM medium.

MNCs (4.5×10⁶) were cultured in IMBM medium for 12 days at 37° C. in a5% CO₂ environment supplemented with the various additives as indicatedin Table 4 below. At 12 days, the cultures were characterized byenumeration and immunophenotypic analysis by flow cytometry performed atNorthwestern Univeristy using a Coulter Flow cytometer. Megakaryocyteswere identified using fluorescently-tagged anti-CD41a (a monoclonalantibody which recognizes gpIIb/IIIa; obtained from Becton Dickinson).

Solution mCRP (prepared as described in Example 1) was added to some ofthe cultures along with 1% or 10% fetal bovine serum (ICN). Theseexperiments were designed to see if mCRP had Megakaryocyte-StimulatingFactor activity (Meg-CSF).

To assess the Megakaryocyte-Potentiation activity (Meg-POT) of mCRP andthe mutant rCRP produced by pIT13 (preparation described in Example 2),aplastic serum or IL-3 (R & D Systems) was added to the cultures as theprimary stimulus. Aplastic serum is the serum of patients with aplasticanemia. The rationale for adding this serum is that such patients havelow levels of blood cells as part of their disease and they must,therefore, be making a lot of "hematopoietic growth factors" to try andcompensate for the low level of cells. Thus, aplastic serum would be asource of naturally-occurring megakaryocyte stimulating factors. Whenused, aplastic serum (AS) was added at 1%, 2.5% or 10%.

The results of the experiments are summarized in Table 4. The resultsare expressed as:

1. total cell number at day 12 of culture (of 4.5×10⁶ seeded);

2. relative frequency of small, intermediate and large CD41+cells;

3. the absolute number of megakaryocytes quantified;

4. the number of megakaryocytes/number seeded cells; and

5. the % of cells remaining alive on day 12, and the % of cells alive onday 12 which are megakaryocytes.

When bone marrow cells were grown for 12 days in the presence of 20μg/ml mCRP, only 7-27% of the starting cell number could be quantifiedas alive on day 12. The percentage of the total cell population whichwas quantified as megakaryocytes on day 12 varied from 0.11% to 0.35%(normal % megakaryocytes is approximately 0.5%). Hence, the addition ofmCRP did not apparently keep the cells alive, nor did it selectivelystimulate the growth of megakaryocytes.

By adding AS at 1%, approximately 50% of the starting cell number wasquantified on day 12. There was a slight increase in the absolute numberof CD41+ cells with AS alone. However, when the MNC's were cultured inthe presence of AS and 20 μg/ml mCRP, the absolute number of CD41+cells, the number of CD41+ cells/million seeded MNC cells, and the % ofCD41+cells/cells alive on day 12 all increased approximately two-fold.This suggests that mCRP augments the action of AS in terms of thedevelopment of human bone marrow cells into the megakaryocyte lineage.

When 2.5% AS was added, again, approximately 50% of the starting cellnumber was quantified as alive on day 12. The absolute number of CD41+cells quantified in AS alone more than doubled (from 6,900 to 16,000),indicating that there was a factor in AS which promoted megakaryocytegrowth. In just AS, the % of cells quantified on day 12 which were CD41+increased from the control level of 0.3% to 0.77%.

When 20 μg/ml or 32 μg/ml mCRP was co-cultured with 2.5% AS, there wasno apparent effect on the survival of the seeded MNCs, but there was anincrease in quantified CD41+ cells above the 16,000 cells quantified in2.5% AS alone. When cultured in the presence of mCRP, 23,000 to 27,000CD41+ cells were quantified (5,100 to 6,000 cells/million seeded MNCs).This represents a 44-69% increase over AS alone. The % of cells alive onday 12 also increased such that 1% to 1.5% of the total cells quantifiedwere now CD41+ cells.

When mutant rCRP was used in place of mCRP, fewer cells were found aliveon day 12 (declining from 40-50% of seeded MNCs found alive on day 12 to29-33% of seeded MNCs). However, of these cells, from 23,000 to 28,000CD41+cells were quantified (5,100 to 6,200 cells/million seeded MNCs),representing from 1.77 to 1.9% of the cells alive on day 12. This datasuggests mCRP and the mutant rCRP enhance the survival and growth ofhuman bone marrow non-adherent mononuclear cells in culture. They alsosuggest that mCRP and mutant rCRP function best as potentiators, ratherthan as primary stimuli, of megakaryocyte growth.

When the MNCs were grown in 10% AS, 78% of the starting cells number wasquantified on day 12. The number of CD41+cells increased to 116,000(25,700/million MNCs seeded on day 1). This represents 3.3% of thosecells still alive on day 12.

When mutant rCRP was co-cultured with MNCs in the presence of 10% AS,the number of cells quantified as alive on day 12 increased to 5.1million, increasing 13% above the starting number of cells seeded. Thissuggests that mutant rCRP promoted cell growth during the 12 days inculture. The number of CD41+ cells quantified increased to 143,000(31,700/million cells seeded; an increase of 23% over 10% AS alone).CD41+ cells accounted for 2.8% of total cells alive.

When IL-3 was used as the primary stimulus instead of AS, the number ofcells alive on day 12 increased from 2.8 million (IL-3 alone; 62.2% ofthe starting cell number) to 3.9 million (IL-3 plus mCRP; 84.8% of thestarting cell number). The absolute numbers of CD41+cells increased19.4%-21.2% in the presence of mCRP compared to IL-3 alone (from 98,000to 117,000 in experiment #1 and from 23,100 to 28,000 in experiment #2).The mutant rCRP also caused an increase in the number of CD41+ cellsquantified in this co-culture (from 23,100 to 28,000).

Modified-CRP and mutant rCRP appear to have megakaryocyte stimulatingactivity in human bone marrow cultures. The effect of mCRP and mutantrCRP appears to be most apparent when used in combination with a primarystimulus of megakaryocyte growth (aplastic serum or IL-3) . In someexperiments mCRP and mutant rCRP appeared to enhance a general growth ofthe mononuclear cell population, with a particular effect onmegakaryocytes (i.e. CD41+ cells).

                                      TABLE 4                                     __________________________________________________________________________    Effect of mCRP on Human Bone Marrow Megakaryocyte Cell Growth                            # of cells alive on day                                                                 % surviving                                                                           Absolute # CD41 +                                                                      # CD41 + cells/10.sup.6                                                                  % CD41 + cells/# cells                                                        alive on                       Treatment 12 cells cells seeded MNC day 12                                  __________________________________________________________________________    None        0.7× 10.sup.6                                                                    15.6    2,450    544        0.35                           + 20 μg/ml sol-mCRP              0.3 × 10.sup.6                                                                     6.7             1,500                                                                      333                                                                            0.11                                                             1% Fetal Bovine Serum                                                                      0.9                                                             × 10.sup.6                                                                   20                                                                       4,500                                                                         1,000                                                                           0.11                         + 20 μg/ml sol-mCRP              0.8 × 10.sup.6                                                                     17.8           4,800                                                                      1,067                                                                           0.11                                                             10% Fetal Bovine Serum                                                                     1.0                                                             × 10.sup.6 22.2                                                         5,500 1,222 0.12                                                               + 20 μg/ml sol-mCRP                                                                   1.2 ×                                                       10.sup.6            26.7                                                                6,600                                                                      1,467 0.12                                                              1% Aplastic Serum                                                                          2.3                                                             × 10.sup.6                                                                   51.1                                                                     6,900                                                                         1,533                                                                           0.3                          + 20 μg/ml sol-mCRP               2.0 × 10.sup.6                                                                    44.4           12,000                                                                     2,667                                                                           0.6                                                              2.5% Aplastic Serum                                                                        2.09                                                            × 10.sup.6  46.4                                                        16,000 3,560  0.77                                                             + 20 μg/ml sol-mCRP                                                                   1.8 ×                                                       10.sup.6           40                                                                  27,000                                                                     6,000                                                                           1.5                    + 32 μg/mlsol-mCRP                2.3 × 10.sup.6                                                                    51.1           23,000                                                                     5,100                                                                             1                                                              + 20 μg/ml rCRP                                                                        1.5 ×                                                      10.sup.6                                                                      33.3           28,000                                                                     6,200                                                                           1.9                                                              + 32 μg/ml rCRP                                                                        1.3 ×                                                      10.sup.6                                                                      28.9           23,000                                                                     5,100                                                                           1.77                                                             10% Aplastic Serum                                                                         3.5                                                             × 10.sup.6 77.8                                                         116,000 25,700 3.3             + 32 μg/ml rCRP                    5.1 × 10.sup.6                                                                   113            143,000                                                                   31,700                                                                           2.8                                                              IL-3 (50 U/ml) (exp #1)                                                                 2.8 ×                                                        10.sup.6 62.2 98,000                                                          21,778 0.48                    + 20 μg/ml  sol-mCRP               3.9 × 10.sup.6                                                                   86.7           117,000                                                                   26,000                                                                           0.67                                                             IL-3 (50 U/ml) (exp #2)                                                                 2.1 ×                                                        10.sup.6                                                                      46.7           23,100                                                                     5,133                                                                           0.24                                                             + 20 μg/ml sol-mCRP                                                                    1.9 ×                                                      10.sup.6                                                                      42.2           9,500                                                                      2,111                                                                           0.11                                                             + 32 μg/ml sol-mCRP                                                                   2.0 ×                                                       10.sup.6                                                                      44.4           28,000                                                                     6,222                                                                           0.31                                                             + 32 μg/ml rCRP                                                                       2.0 ×                                                       10.sup.6                                                                      44.4           28,000                                                                     6,222                                                                           0.31           __________________________________________________________________________     4.5 × 10.sup.6 human bone marrow nonadherent mononuclear cells          seeded on day 1                                                          

Example 10 Testing of Mutant rCRP In Mice

Three experiments were performed. In all three experiments, groups offive female BALB/c mice (Harlan-Sprague-Dawley) were used.

A. Experiment 1

In the first, the mice were injected intravenously with the mutant rCRPproduced by pIT13 (preparation described in Example 2), Monday throughFriday for two consecutive weeks. The mutant rCRP was injected at 1mg/kg/dose, 3 mg/kg/dose or 10 mg/kg/dose. Blood samples were drawn ondays 1 (just before the first injection), 4, 8, 11, 15, 18 and 23.Platelet counts were quantified using a Sysmex F-800 cell counter (ToaMedical Electronics, Japan).

FIGS. 17A-C illustrate the results of this experiment. All three dosesof intravenously injected mutant rCRP caused a transientthrombocytopoenia which was noted on day 4. The platelet drop on thisday was found to be significantly different in mice treated with thehighest dose (10 mg/kg/dose) of mutant rCRP compared to the meanplatelet level noted in animals receiving buffer control (FIG. 1C;p<0.05). The mean platelet levels on all subsequent sample days in allmutant rCRP dose groups then increased to or above the mean plateletlevel of the control group of animals on each study day. Statisticallysignificant increases were noted in all three dose groups. Of note, the1 mg/kg/dose group of animals showed statistical significance overcontrol on study day 8 (FIG. 17A), the 3 mg/kg/dose group of animalsshowed statistical significance over control on study day 12 (FIG. 17B),and the 10 mg/kg/dose group of animals showed statistical significanceover control on study day 15 (FIG. 17C). Data are plotted as mean±standard error. Only p values of ≦0.05 are considered statisticallysignificant.

B. Experiment 2

In the second experiment, mutant rCRP produced by pIT13 was injectedsubcutaneously instead of intravenously in the same amounts and over thesame time frame as in experiment 1. Blood samples were drawn on days 1,4, 8, 16, 20 and 24. Platelet counts were quantified using a SysmexF-800 cell counter.

FIGS. 18A-C illustrate the results of this experiment. Thethrombocytopoenia noted in experiment 1 was not apparent using thesubcutaneous route of injection, and increases in peripheral bloodplatelet levels were again observed in all three dose groups during theexperiment and within 8 days after the last injection. The same trend ofstatistically significant increases seen in the first experiment wasnoted in the second experiment, again being correlated with increasingmutant rCRP dose amounts. That is, the 1 mg/kg/dose group of animalsshowed a statistically significant increase over the control mean onstudy day 4 (FIG. 18A), the 3 mg/kg/dose group of animals showed astatistically significant increase over the control mean on study day 12(FIG. 18B), and the 10 mg/kg/dose group of animals showed astatistically significant increase over the control mean on study day 20(FIG. 18C).

C. Experiment 3

In the third experiment, the effect of multiple injections of mutantrCRP produced by pIT13 at the 3 mg/kg/dose level was compared to theeffect of a similarly administered amount of the solution mCRP.Subcutaneous injections were performed done on days 1 through 4, and 8through 16. Blood samples were drawn and analyzed for platelet counts ondays 1, 4, 8, 12, 16, 20 and 24.

FIG. 19 shows that the effect of multiple subcutaneous injections ofmutant rCRP and mCRP on increasing peripheral blood platelet counts inmice are similar. Platelet counts for both mutant rCRP and mCRP on studydays 8, 12 and 16 (i.e., days on which injections were given) werestatistically elevated to p values at least <0.02 (indicated by *), overthe day 1 mean platelet counts in each group of animals. The increasedplatelet count persisted in mCRP-treated animals on day 20 (four daysafter the last injection). In both mutant rCRP-treated and mCRP-treatedanimals, the peripheral blood platelet values eventually dropped towardpre-injection levels after therapy was stopped.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 24                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 28                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer         - - <400> SEQUENCE: 1                                                         - - gggccatatg cagacagaca tgtcgagg         - #                  - #                 28                                                                      - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 2                                                         - - gaagcgccac agtgaaggct            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 16                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 3                                                         - - cactgtggcg ctccac             - #                  - #                      - #    16                                                                   - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 4                                                         - - ttgtcgcgat gtgtactgg             - #                  - #                      - # 19                                                                   - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 17                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 5                                                         - - cacatcgcga caagctg             - #                  - #                      - #   17                                                                   - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 6                                                         - - ggcgagatct gaggtacctt cagg          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 12                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 7                                                         - - tttggccaga ca              - #                  - #                      - #       12                                                                   - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 15                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:plasmid         - - <400> SEQUENCE: 8                                                         - - catatggcta gcatg              - #                  - #                      - #    15                                                                   - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 9                                                         - - gcttttggcc agacagacat g           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:mutant          - - <400> SEQUENCE: 10                                                        - - catatggcta gccagacaga catg          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 11                                                        - - Leu Ser Ser Thr Arg Gly Tyr Ser Ile Phe                                    1               5 - #                 10                                     - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:mutant          - - <400> SEQUENCE: 12                                                        - - Ser Tyr Phe Gly Leu Arg Ile                                                1               5                                                            - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 13                                                        - - Thr Arg Gly Tyr Ser Ile Phe Ser Tyr Ala Th - #r Lys Arg Gln Asp Asn        1               5 - #                 10 - #                 15              - - Glu Ile Leu Ile Phe Trp Ser Lys Asp Ile Gl - #y                                       20     - #             25                                         - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 81                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 14                                                        - - acccgtgggt acagtatttt ctcgtatgcc accaagagac aagacaatga ga -             #ttctcata     60                                                                 - - ttttggtcta aggatatagg a           - #                  - #                      - #81                                                                  - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:hypothetic    al                                                                              - - <400> SEQUENCE: 15                                                        - - Thr Arg Gly Thr Val Phe Ser Arg Met Pro Pr - #o Arg Asp Lys Thr Met        1               5 - #                 10 - #                 15              - - Arg Phe Ser Tyr Phe Gly Leu Arg Ile                                                   20     - #             25                                         - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 80                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:hypothetic    al                                                                              - - <400> SEQUENCE: 16                                                        - - acccggggta cagtattttc tcgtatgcca ccaagagaca agacaatgag at -             #tctcatat     60                                                                 - - tttggtctaa ggatatagga            - #                  - #                      - # 80                                                                  - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 17                                                        - - Thr Arg Gly Thr Val Phe Ser Arg Met Pro Pr - #o Arg Asp Lys Thr Met        1               5 - #                 10 - #                 15              - - Arg Phe Phe Ile Phe Trp Ser Lys Asp Ile Gl - #y                                       20     - #             25                                         - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 81                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 18                                                        - - acccggggta cagtattttc tcgtatgcca ccaagagaca agacaatgag at -             #tcttcata     60                                                                 - - ttttggtcta aggatatagg a           - #                  - #                      - #81                                                                  - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 28                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:mutant          - - <400> SEQUENCE: 19                                                        - - cctcgacccg gggtacagta ttttctcg         - #                  - #                 28                                                                      - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 20                                                        - - cctcgacccg tggttacagc attttctcg         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 21                                                        - - cccgcgaaat taatacgact cacta          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 22                                                        - - cgagaaaatg ctgtaaccac gggtcgagg         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 23                                                        - - ctttgttagc agccggatcc gaggta          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:mutant          - - <400> SEQUENCE: 24                                                        - - Arg Gly Thr Val Phe Ser Arg Met Pro Pro Ar - #g Asp Lys Thr Met Arg        1               5 - #                 10 - #                 15              - - Phe Ser Tyr Phe Gly Leu Lys                                                           20                                                              __________________________________________________________________________

What is claimed is:
 1. A method of stimulating thrombocytopoiesis in amammal comprising administering to the mammal an effective amount ofmutant CRP subunit or preCRP protein expressing neo-CRP antigenicity ina pharmaceutically-acceptable carrier, said mutant protein having atleast one cysteine deleted from or replaced by another amino acid ascompared with an unmutated CRP subunit or preCRP protein.
 2. The methodof claim 1 wherein the mutant protein is administered to the mammal bysubcutaneous injection.
 3. The method of claim 1 wherein about 3milligrams per kilogram body weight of the mutant protein isadministered to the mammal.
 4. The method of claim 1 wherein the mutantprotein is administered to the mammal prior to administeringchemotherapy to the mammal.
 5. The method of claim 1 wherein the mutantprotein is administered to the mammal prior to administering sub-lethalor lethal doses of irradiation to the mammal.
 6. The method of claim 1wherein the mutant protein has all of the cysteines deleted or replaced.7. The method of claim 1 wherein the mutant protein has at least onecysteine replaced by alanine.
 8. The method of claim 7 wherein themutant protein has all of the cysteines replaced by alanines.
 9. Amethod of treating thrombocytopenia in a mammal comprising administeringto a mammal having thrombocytopenia an effective amount of mutant CRPsubunit or preCRP protein expressing neo-CRP antigenicity in apharmaceutically-acceptable carrier, said mutant having at least onecysteine deleted from or replaced by another amino acid as compared withan unmutated CRP subunit or PCRP protein.
 10. The method of claim 9wherein the mammal having thrombocytopenia has a thrombocyte level below100,000 per cubic millimeter peripheral blood prior to administration ofthe mutant protein.
 11. The method of claim 9 wherein two or moresubcutaneous injections of the mutant protein are administered to themammal.
 12. The method of claim 9 wherein the mutant protein has all ofthe cysteines deleted or replaced.
 13. The method of claim 9 wherein themutant protein has at least one cysteine replaced by alanine.
 14. Themethod of claim 9 wherein the mutant protein has all of the cysteinesreplaced by alanines.
 15. The method of claim 1, wherein the mutantprotein has glutamine substituted for a fomylated-methionine at theN-terminal end, and alanine substituted for cysteine at amino acidpositions 36 and
 97. 16. The method of treating thrombocytopenia ofclaim 9, wherein the mutant protein has glutamine substituted for afomylated-methionine at the N-terminal end, and alanine substituted forcysteine at amino acid positions 36 and
 97. 17. The method of claim 1,wherein the mutant protein has the amino acid sequence at positions47-69 as shown in SEQ ID NO:24.
 18. The method of treating of claim 9,wherein the mutant protein has the amino acid sequence at positions47-69 as shown in SEQ ID NO:24.